Title: Non-neuronal Cholinergic Muscarinic Acetylcholine Receptors in the Regulation of Immune Function Mashimo M, Kawashima K, Fujii T Ref: Biol Pharm Bull, 45:675, 2022 : PubMed
Immune cells such as T and B cells, monocytes and macrophages all express most of the cholinergic components of the nervous system, including acetylcholine (ACh), choline acetyltransferase (ChAT), high affinity choline transporter, muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively), and acetylcholinesterase (AChE). Because of its efficient cleavage by AChE, ACh synthesized and released from immune cells acts only locally in an autocrine and/or paracrine fashion at mAChRs and nAChRs on themselves and other immune cells located in close proximity, leading to modification of immune function. Immune cells generally express all five mAChR subtypes (M(1)-M(5)) and neuron type nAChR subunits alpha2-alpha7, alpha9, alpha10, beta2-beta4. The expression pattern and levels of mAChR subtypes and nAChR subunits vary depending on the tissue involved and its immunological status. Immunological activation of T cells via T-cell receptor-mediated pathways and cell adhesion molecules upregulates ChAT expression, which facilitates the synthesis and release of ACh. At present, alpha7 nAChRs expressed in macrophages are receiving much attention because they play a central role in anti-inflammatory cholinergic pathways. However, it now appears that through modification of cytokine synthesis, G(q/11)-coupled mAChRs play a prominent role in regulation of T cell proliferation and differentiation and B cell immunoglobulin class switching. It is anticipated that greater understanding of G(q/11)-coupled mAChRs on immune cells will provide an opportunity to develop new and effective treatments for immunological disorders.
Title: Regulation of Immune Functions by Non-Neuronal Acetylcholine (ACh) via Muscarinic and Nicotinic ACh Receptors Mashimo M, Moriwaki Y, Misawa H, Kawashima K, Fujii T Ref: Int J Mol Sci, 22:, 2021 : PubMed
Acetylcholine (ACh) is the classical neurotransmitter in the cholinergic nervous system. However, ACh is now known to regulate various immune cell functions. In fact, T cells, B cells, and macrophages all express components of the cholinergic system, including ACh, muscarinic, and nicotinic ACh receptors (mAChRs and nAChRs), choline acetyltransferase, acetylcholinesterase, and choline transporters. In this review, we will discuss the actions of ACh in the immune system. We will first briefly describe the mechanisms by which ACh is stored in and released from immune cells. We will then address Ca(2+) signaling pathways activated via mAChRs and nAChRs on T cells and B cells, highlighting the importance of ACh for the function of T cells, B cells, and macrophages, as well as its impact on innate and acquired (cellular and humoral) immunity. Lastly, we will discuss the effects of two peptide ligands, secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) and hippocampal cholinergic neurostimulating peptide (HCNP), on cholinergic activity in T cells. Overall, we stress the fact that ACh does not function only as a neurotransmitter; it impacts immunity by exerting diverse effects on immune cells via mAChRs and nAChRs.
PURPOSE: To investigate whether proximal subtotal pancreatectomy (PSTP) is superior to total pancreatectomy (TP) for preserving postoperative endocrine function, and to identify the pre-operative risk factors influencing prognosis after TP and PSTP. METHODS: The subjects of this retrospective study were patients who underwent TP (n = 15) or PSTP (n = 16) between 2008 and 2018 in our hospital. First, we compared the incidence of hypoglycemia within 30 days after surgery and the total daily amount of insulin needed in the 30 days after TP vs. PSTP. Then, we compared the prognoses between the groups. RESULTS: The incidence of hypoglycemia in the 30 days after surgery was significantly lower in the PSTP group than in the TP group (n = 0 vs. n = 5; p < 0.001). The total amount of daily insulin given was also significantly lower after PSTP than after TP: (0 units vs. 18 units, p = 0.001). Lower lymphocyte counts (p = 0.014), lower cholinesterase (p = 0.021), and lower prognostic nutrition index (p = 0.021) were identified as significant risk factors for hypoglycemia in the TP group. Low cholinesterase (p = 0.015) and a low prognostic nutrition index (p = 0.048) were significantly associated with an unfavorable prognosis in the TP group, but not in the PSTP group. CONCLUSIONS: PSTP may be a feasible alternative to TP to preserve endocrine function, especially for malnourished patients.
T and B cells, macrophages and dendritic cells (DCs) all express most of the components necessary for a functional cholinergic system. This includes choline acetyltransferase (ChAT), muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs, respectively) and acetylcholinesterase (AChE). Immunological activation of T cells up-regulates cholinergic activity, including ChAT and AChE expression. Moreover, toll-like receptor agonists induce ChAT expression in DCs and macrophages, suggesting cholinergic involvement in the regulation of immune function. Immune cells express all five M1-M5 mAChR subtypes and several nAChR subtypes, including alpha7. Modulation of antigen-specific antibody and pro-inflammatory cytokine production in M1/M5 mAChR gene-knockout (KO) and alpha7 nAChR-KO mice further support the idea of a non-neuronal cholinergic system contributing to the regulation of immune function. Evidence also suggests that alpha7 nAChRs are involved in suppressing DC and macrophage activity, leading to suppression of T cell differentiation into effector T cells. These findings suggest the possibility that immune function could be modulated by manipulating immune cell cholinergic activity using specific agonists and antagonists. Therefore, a fuller understanding of the immune cell cholinergic system should be useful for the development of drugs and therapeutic strategies for the treatment of inflammation-related diseases and cancers.
T and B cells express most cholinergic system components-e.g., acetylcholine (ACh), choline acetyltransferase (ChAT), acetylcholinesterase, and both muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively). Using ChATBAC-eGFP transgenic mice, ChAT expression has been confirmed in T and B cells, dendritic cells, and macrophages. Moreover, T cell activation via T-cell receptor/CD3-mediated pathways upregulates ChAT mRNA expression and ACh synthesis, suggesting that this lymphocytic cholinergic system contributes to the regulation of immune function. Immune cells express all five mAChRs (M1-M5). Combined M1/M5 mAChR-deficient (M1/M5-KO) mice produce less antigen-specific antibody than wild-type (WT) mice. Furthermore, spleen cells in M1/M5-KO mice produce less tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, suggesting M1/M5 mAChRs are involved in regulating pro-inflammatory cytokine and antibody production. Immune cells also frequently express the alpha2, alpha5, alpha6, alpha7, alpha9, and alpha10 nAChR subunits. alpha7 nAChR-deficient (alpha7-KO) mice produce more antigen-specific antibody than WT mice, and spleen cells from alpha7-KO mice produce more TNF-alpha and IL-6 than WT cells. This suggests that alpha7 nAChRs are involved in regulating cytokine production and thus modulate antibody production. Evidence also indicates that nicotine modulates immune responses by altering cytokine production and that alpha7 nAChR signaling contributes to immunomodulation through modification of T cell differentiation. Together, these findings suggest the involvement of both mAChRs and nAChRs in the regulation of immune function. The observation that vagus nerve stimulation protects mice from lethal endotoxin shock led to the notion of a cholinergic anti-inflammatory reflex pathway, and the spleen is an essential component of this anti-inflammatory reflex. Because the spleen lacks direct vagus innervation, it has been postulated that ACh synthesized by a subset of CD4+ T cells relays vagal nerve signals to alpha7 nAChRs on splenic macrophages, which downregulates TNF-alpha synthesis and release, thereby modulating inflammatory responses. However, because the spleen is innervated solely by the noradrenergic splenic nerve, confirmation of an anti-inflammatory reflex pathway involving the spleen requires several more hypotheses to be addressed. We will review and discuss these issues in the context of the cholinergic system in immune cells.
Title: 5-Bromoindirubin 3'-(O-oxiran-2-ylmethyl)oxime: A long-acting anticancer agent and a suicide inhibitor for epoxide hydrolase Ichimaru Y, Fujii T, Saito H, Sano M, Uchiyama T, Miyairi S Ref: Bioorganic & Medicinal Chemistry, 25:4665, 2017 : PubMed
Indirubin 3'-oxime (Indox (1b)) suppresses cancer cell growth (IC50: 15muM towards HepG2 cells) and inhibits cell cycle-related kinases such as cyclin-dependent kinases and glycogen synthase kinase-3beta. We have previously reported that the conjugation of 1b with oxirane, a protein-reactive component, enhanced the cytotoxic activity of Indox as determined from the IC50 value (1.7muM) of indirubin 3'-(O-oxiran-2-ylmethyl)oxime (Epox/Ind (1c)). Here we prepared Epox/Ind derivatives with one or two halogen atoms or a methoxy group on the aromatic ring(s) of an Indox moiety and studied the structure-activity relationships of the substituent(s). We found that bromine-substitution at the 5-position on 1c or any Epox/Ind derivative(s) having bromine on the aromatic ring except Epox/6'-Br-Ind was efficient to improving anticancer activity. Of the 22 Epox/Ind derivatives, 5-bromoindirubin 3'-(O-oxiran-2-ylmethyl)oxime (Epox/5-Br-Ind (2c)) was the best anticancer agent in both short- (24h) (IC50: 0.67muM) and extended-duration (72h) cultures. The high anticancer activity of 2c was partly due to it being a poor substrate and a suicide inhibitor for epoxide hydrolase as epoxide hydrolase was identified as the enzyme primarily responsible for the metabolism of 2c.
Title: Re-characterization of mono-2-ethylhexyl phthalate hydrolase belonging to the serine hydrolase family Iwata M, Imaoka T, Nishiyama T, Fujii T Ref: J Biosci Bioeng, 122:140, 2016 : PubMed
A novel bacterium assimilating di-2-ethylhexyl phthalate as a sole carbon source was isolated, and identified as a Rhodococcus species and the strain was named EG-5. The strain has a mono-2-ethylhexyl phthalate (MEHP) hydrolase (EG-5 MehpH), which exhibits some different enzymatic features when compared with the previously reported MEHP hydrolase (P8219 MehpH) from Gordonia sp. These differences include different pH optimum activity, maximal reaction temperature and heat stability. The Km and Vmax values of EG-5 MehpH were significantly higher than those of P8219 MehpH. The primary structure of EG-5 MehpH showed the highest sequence identity to that of P8219 MehpH (39%) among hydrolases. The phylogenetic tree suggested that EG-5 MehpH and P8219 MehpH were categorized in different groups of the novel MEHP hydrolase family. Mutation of a conserved R(109) residue of EG-5 MehpH to a hydrophobic residue resulted in a dramatic reduction in the Vmax value towards MEHP without affecting the Km value. These results indicate that this residue may neutralize the negative charge of a carboxylate anion of MEHP, and thus inhibit the catalytic nucleophile from attacking the ester bond. In other words, the R residue blocks inhibition from the carboxylate anion of MEHP. Recently, registered hypothetical proteins exhibiting 98% or 99% identities for EG-5 MehpH or for P8219 MehpH were found from some pathogens belonging to Actinomycetes. The protein may have other activities besides MEHP hydrolysis and function in other physiological reactions in some Actinomycetes.
In 1929, Dale and Dudley described the first reported natural occurrence of acetylcholine (ACh) in an animal's body. They identified this ACh in the spleens of horses and oxen, which we now know suggests possible involvement of ACh in the regulation of lymphocyte activity and immune function. However, the source and function of splenic ACh were left unexplored for several decades. Recent studies on the source of ACh in the blood revealed ACh synthesis catalyzed by choline acetyltransferase (ChAT) in CD4(+) T cells. T and B cells, macrophages and dendritic cells (DCs) all express all five muscarinic ACh receptor subtypes (mAChRs) and several subtypes of nicotinic AChRs (nAChRs), including alpha7 nAChRs. Stimulation of these mAChRs and nAChRs by their respective agonists causes functional and biochemical changes in the cells. Using AChR knockout mice, we found that M(1)/M(5) mAChR signaling up-regulates IgG(1) and pro-inflammatory cytokine production, while alpha7 nAChR signaling has the opposite effect. These findings suggest that ACh synthesized by T cells acts in an autocrine/paracrine fashion at AChRs on various immune cells to modulate immune function. In addition, an endogenous allosteric and/or orthosteric alpha7 nAChR ligand, SLURP-1, facilitates functional development of T cells and increases ACh synthesis via up-regulation of ChAT mRNA expression. SLURP-1 is expressed in CD205(+) DCs residing in the tonsil in close proximity to T cells, macrophages and B cells. Collectively, these findings suggest that ACh released from T cells along with SLURP-1 regulates cytokine production by activating alpha7 nAChRs on various immune cells, thereby facilitating T cell development and/or differentiation, leading to immune modulation.
Title: SLURP-1, an endogenous alpha7 nicotinic acetylcholine receptor allosteric ligand, is expressed in CD205(+) dendritic cells in human tonsils and potentiates lymphocytic cholinergic activity Fujii T, Horiguchi K, Sunaga H, Moriwaki Y, Misawa H, Kasahara T, Tsuji S, Kawashima K Ref: Journal of Neuroimmunology, 267:43, 2014 : PubMed
Immune cells often express various nicotinic ACh receptor (nAChR) subtypes, including alpha7 nAChRs, as well as mRNA encoding secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide (SLURP)-1, an endogenous alpha7 nAChR allosteric ligand. We detected SLURP-1 immunoreactivity in CD205(+) dendritic cells (DCs) residing in human tonsils. Phytohemagglutinin (PHA, 10mug/ml), a T cell activator, attenuated cell proliferation and increased the ACh content of MOLT-3 human leukemic T cells compared with the vehicle control. Methyllycaconitine (MLA, 100nM), a specific alpha7 nAChR antagonist, abolished all effects elicited by PHA. Recombinant (r)SLURP-1 (0.5mug/ml) attenuated peripheral blood mononuclear cell proliferation and increased ChAT gene expression and the ACh content in MOLT-3 cells compared with the control, all of which were abolished by MLA. This suggests SLURP-1 activates cholinergic transmission by potentiating ACh synthesis and its action at alpha7 nAChRs, thereby facilitating functional development of T cells. These findings support the notion that SLURP-1 acts as a key modulator of immune responses.
Paraphoma-related fungal strain B47-9 secreted a biodegradable plastic (BP)-degrading enzyme which amounted to 68 % (w/w) of the total secreted proteins in a culture medium containing emulsified poly(butylene succinate-co-adipate) (PBSA) as sole carbon source. The gene for this enzyme was found to be composed of an open reading frame consisting of 681 nucleotides encoding 227 amino acids and two introns. Southern blot analysis showed that this gene exists as a single copy. The deduced amino acid sequence suggested that this enzyme belongs to the cutinase (E.C.3.1.1.74) family; thus, it was named P araphoma-related fungus cutinase-like enzyme (PCLE). It degraded various types of BP films, such as poly(butylene succinate), PBSA, poly(butylene adipate-co-terephthalate), poly(epsilon-caprolactone), and poly(DL-lactic acid). It has a molecular mass of 19.7 kDa, and an optimum pH and temperature for degradation of emulsified PBSA of 7.2 and 45 degrees C, respectively. Ca(2+) ion at a concentration of about 1.0 mM markedly enhanced the degradation of emulsified PBSA.
There is a need to speed up the degradation of used agricultural mulch films that are made of biodegradable plastics (BPs) in the field. Treating them with BP-degrading enzymes could be a solution to this problem. A cutinase-like enzyme of yeast Pseudozyma antarctica (PaE) has wide specificity of BPs degradation, but needs to be produced efficiently. Here we report that the production of PaE by P. antarctica can be increased by using xylose as carbon source. BP-degradation activity was analyzed using a polybutylene succinate-co-adipate (PBSA) emulsion as the substrate. Strain P. antarctica GB-4(1)W was found to be the best PaE producer among the tested strains. Using a 5-L jar fermentor with xylose fed-batch cultivation, high PaE productivity could be maintained and about 21 U/ml of PaE was obtained in 120 h. This amount was 100 times higher than the amount that we obtained previously (0.21 U/ml by flask cultivation using glycerol as carbon source). Under repeated xylose fed-batch cultivation with 24 h intervals, the maximum PaE production rate (0.34 U/ml/h) was maintained and the highest PaE productivity (28,000 U/2 L/d) was repeatedly obtained for 7 intervals. The activity of filtered jar-culture (crude PaE) was stable over 12 weeks at 4 degrees C. Commercially available BP mulch films (20 mum thickness, cut into 1-cm-squares) were completely degraded by submerging them in crude PaE (2 U/ml) at 30 degrees C in 24 h. These results indicated that concentrated PaE can rapidly degrade the strength of BP mulch films in the field so that they do not interfere with plowing.
Pseudozyma antarctica JCM 10317 exhibits a strong degradation activity for biodegradable plastics (BPs) such as agricultural mulch films composed of poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA). An enzyme named PaE was isolated and the gene encoding PaE was cloned from the strain by functional complementation in Saccharomyces cerevisiae. The deduced amino acid sequence of PaE contains 198 amino acids with a predicted molecular weight of 20,362.41. High identity was observed between this sequence and that of cutinase-like enzymes (CLEs) (61-68%); therefore, the gene encoding PaE was named PaCLE1. The specific activity of PaE against emulsified PBSA was 54.8+/-6.3 U/mg. In addition to emulsified BPs, PaE degraded solid films of PBS, PBSA, poly(epsilon-caprolactone), and poly(lactic acid).
Two yeast strains, which have the ability to degrade biodegradable plastic films, were isolated from the larval midgut of a stag beetle, Aegus laevicollis. Both of them are most closely related to Cryptococcus magnus and could degrade biodegradable plastic (BP) films made of poly(butylene succinate) (PBS) and poly(butylene succinate-co-adipate) (PBSA) effectively. A BP-degrading enzyme was purified from the culture broth of one of the isolated strains employing a newly developed affinity purification method based on the binding action of the enzyme to the substrate (emulsified PBSA) and its subsequent degradative action toward the substrate. Partial amino acid sequences of this enzyme suggested that it belongs to the cutinase family, and thus, the enzyme was named CmCut1. It has a molecular mass of 21 kDa and a degradative activity for emulsified PBSA which was significantly enhanced by the simultaneous presence of Ca(2+) or Mg(2+) at a concentration of about 2.5 mM. Its optimal pH was 7.5, and the optimal temperature was 40 degrees C. It showed a broad substrate specificity for p-nitrophenyl (pNP)-fatty acid esters ranging from pNP-acetate (C2) to pNP-stearate (C18) and films of PBSA, PBS, poly(epsilon-caprolactone), and poly(lactic acid).
AIMS: Muscarinic and nicotinic acetylcholine (ACh) receptors are expressed in immune cells. ACh synthesized by choline acetyltransferase (ChAT) and released in T cells binds to these receptors. Furthermore, we have recently demonstrated the involvement of mediatophore, a homooligomer of a 16-kDa proteolipid subunit of vacuolar H(+)-ATPase, in ACh release from T cells. In this study, we investigated the effects of phorbol 12-myristate 13-acetate (PMA), dibutyryl cAMP (dbcAMP) and FK506, an immunosuppressant calcineurin inhibitor, on lymphocytic cholinergic activity in T cells. MAIN METHODS: We determined the content and release of ACh in human leukemic T cell line MOLT-3 cells using a sensitive and specific radioimmunoassay for ACh. In addition, expression of ChAT mRNA and ChAT activity were investigated using reverse-transcription-polymerase chain reaction and Fonnum method, respectively. KEY FINDINGS: Phytohemagglutinin (PHA), a T-cell activator, up-regulated ChAT mRNA expression, synthesis and release of ACh. PMA, a protein kinase C (PKC) activator, and dbcAMP, a protein kinase A (PKA) activator, also increased ChAT activity and ACh synthesis by up-regulating ChAT gene expression. FK506 inhibited PHA-induced up-regulation of ChAT mRNA expression, suggesting the involvement of calcineurin-mediated pathways in ChAT gene transcription. SIGNIFICANCE: Activation of PKC and PKA up-regulates ACh synthesis in T cells, and immunological activation triggers ChAT gene transcription through calcineurin-mediated pathways.
Immunological stimulation of T cells by phytohemagglutinin (PHA) enhances the synthesis and release of acetylcholine (ACh), suggesting a role for the lymphocytic cholinergic system in the regulation of immune function. In the present study, we used two human leukemic T cell lines as models to investigate whether mediatophore, a homooligomer of a 16-kDa subunit homologous to the proteolipid subunit c of vacuolar H(+)-ATPase (V-ATPase), is involved in mediating ACh release from T cells. Immunohistochemical analysis revealed the presence of mediatophore in the cytoplasm and on the plasma membrane of both T cell lines. Mediatophore gene expression was up-regulated by immunological T cell activation by PHA. Transfection of anti-mediatophore small interference RNA down-regulated mediatophore gene expression and significantly reduced ACh release. These results suggest that T cells express mediatophore, which then plays a key role in mediating ACh release, and that mediatophore expression is regulated by immunological stimulation.
Anaerobic ammonium oxidation (anammox) and denitrification are two distinct microbial reactions relevant to the global nitrogen cycle. The proposed initial step of the anammox reactions, reduction of nitrite to nitric oxide, has been postulated to be identical to that in denitrification catalyzed by the dissimilatory nitrite reductase of the cytochrome cd(1)-type. Here, we characterized the copper-containing nitrite reductase homolog encoded by nirK detected in the genome of an anammox bacterium strain KSU-1. We hypothesize that this NirK-type nitrite reductase, rather than a nitrite reductase of the cytochrome cd(1)-type (NirS), is likely to catalyze nitrite reduction in anammox organism KSU-1.
BACKGROUND: Although donepezil, an acetylcholinesterase inhibitor, has been proved to be effective in ameliorating cognitive impairment in Parkinson's disease with dementia (PDD), the responsiveness of patients to donepezil therapy varies. [5-(11)C-methoxy]donepezil, the radiolabeled form of donepezil, is a ligand for positron emission tomography (PET), which can be exploited for the quantitative analysis of donepezil binding to acetylcholinesterase and for cholinergic imaging. OBJECTIVES: To investigate the deficits of the cholinergic system in the brain in PDD and its association with response to donepezil therapy. METHODS: Twelve patients with PDD and 13 normal control subjects underwent [5-(11)C-methoxy]donepezil-PET imaging. For patients with PDD, daily administration of donepezil was started after [5-(11)C-methoxy]donepezil-PET imaging and continued for 3 months. RESULTS: In the PDD group, the mean total distribution volume of the cerebral cortices was 22.7% lower than that of the normal control group. The mean total distribution volume of the patients with PDD was significantly correlated with improvement of visuoperceptual function after 3 months of donepezil therapy. CONCLUSION: The results suggest that donepezil therapy is more effective in patients with less decrease in acetylcholinesterase, a binding site of donepezil, at least in the specific cognitive domain.
Title: Critical roles of acetylcholine and the muscarinic and nicotinic acetylcholine receptors in the regulation of immune function Kawashima K, Fujii T, Moriwaki Y, Misawa H Ref: Life Sciences, 91:1027, 2012 : PubMed
Lymphocytes express both muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs, respectively), and stimulation of mAChRs and nAChRs produces various biochemical and functional changes. Although it has been postulated that parasympathetic cholinergic nerves directly innervate immune cells, no evidence has supported this hypothesis. We measured ACh in the blood of various animal species and determined its localization in T cells using a sensitive and specific radioimmunoassay. Furthermore, we showed that T cells express choline acetyltransferase (ChAT), an ACh synthesizing enzyme. Immunological T cell activation enhances ACh synthesis through the up-regulation of ChAT expression, suggesting lymphocytic cholinergic activity is related to immunological activity. Most immune cells such as T cells, B cells, and monocytes express all five subtypes of mAChRs (M(1)-M(5)), and various subunits of the nAChR, such as alpha3, alpha5, alpha7, alpha9, and alpha10. Studies on serum antibody production in M(1) and M(5) combined mAChR gene knockout (KO) mice immunized with ovalbumin (OVA) revealed that M(1)/M(5) mAChRs up-regulate TNF-alpha, IFN-gamma and IL-6 production in spleen cells, leading to an elevation of serum anti-OVA specific IgG(1). In contrast, studies of nAChR alpha7 subunit gene KO mice immunized with OVA show that alpha7 nAChRs down-regulate these proinflammatory cytokines, thereby leading to a reduction of anti-OVA specific IgG(1). Taken together, these findings demonstrate that both mAChRs and nAChRs modulate production of cytokines, such as TNF-alpha, resulting in a modification of antibody production. These findings support the notion that a non-neuronal cholinergic system is involved in the regulation of immune cell function.
Title: Reconciling neuronally and nonneuronally derived acetylcholine in the regulation of immune function Kawashima K, Fujii T, Moriwaki Y, Misawa H, Horiguchi K Ref: Annals of the New York Academy of Sciences, 1261:7, 2012 : PubMed
Immune cells, including lymphocytes, express muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs, respectively), and agonist stimulation of these AChRs causes functional and biochemical changes in the cells. The origin of the ACh that acts on immune cell AChRs has remained unclear until recently, however. In 1995, we identified choline acetyltransferase mRNA and protein in human T cells, and found that immunological T cell activation potentiated lymphocytic cholinergic transmission by increasing ACh synthesis and AChR expression. We also found that M(1) /M(5) mAChR signaling upregulates IgG(1) and proinflammatory cytokine production, whereas alpha7 nAChR signaling has the opposite effect. These findings suggest that ACh synthesized by T cells acts as an autocrine and/or paracrine factor via AChRs on immune cells to modulate immune function. In addition, a recently discovered endogenous allosteric alpha7 nAChR ligand, SLURP-1, also appears to be involved in modulating normal T cell function.
Alphaproteobacterium strain Q-1 is able to oxidize iodide (I(-)) to molecular iodine (I(2)) by an oxidase-like enzyme. One of the two isoforms of the iodide-oxidizing enzyme (IOE-II) produced by this strain was excised from a native polyacrylamide gel, eluted, and purified. IOE-II appeared as a single band (51 kDa) and showed significant in-gel iodide-oxidizing activity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis without heat treatment. However, at least two bands with much higher molecular masses (150 and 230 kDa) were observed with heat treatment (95 degrees C, 3 min). IOE-II was inhibited by NaN(3), KCN, EDTA, and a copper chelator, o-phenanthroline. In addition to iodide, IOE-II showed significant activities toward phenolic compounds such as syringaldazine, 2,6-dimethoxy phenol, and p-phenylenediamine. IOE-II contained copper atoms as prosthetic groups and had UV/VIS absorption peaks at 320 and 590 nm. Comparison of several internal amino acid sequences obtained from trypsin-digested IOE-II with a draft genome sequence of strain Q-1 revealed that the products of two open reading frames (IoxA and IoxC), with predicted molecular masses of 62 and 71 kDa, are involved in iodide oxidation. Furthermore, subsequent tandem mass spectrometric analysis repeatedly detected peptides from IoxA and IoxC with high sequence coverage (32 to 40%). IoxA showed homology with the family of multicopper oxidases and included four copper-binding regions that are highly conserved among various multicopper oxidases. These results suggest that IOE-II is a multicopper oxidase and that it may occur as a multimeric complex in which at least two proteins (IoxA and IoxC) are associated.
Title: Mechanisms of chronic nicotine treatment-induced enhancement of the sensitivity of cortical neurons to the neuroprotective effect of donepezil in cortical neurons Takada-Takatori Y, Kume T, Izumi Y, Niidome T, Fujii T, Sugimoto H, Akaike A Ref: J Pharmacol Sci, 112:265, 2010 : PubMed
We have previously shown that chronic donepezil treatment induces nicotinic acetylcholine receptor up-regulation and enhances the sensitivity of the neurons to the neuroprotective effect of donepezil. Further analyses revealed that the nicotinic receptor is involved in this enhancement. In this study, we examined whether nicotinic receptor stimulation is sufficient to make neurons more sensitive to donepezil. We treated primary cultures of rat cortical neurons with nicotine and confirmed that chronic nicotine treatment induced nicotinic receptor up-regulation and made the neurons more sensitive to the neuroprotective effects of donepezil. Analyses with receptor antagonists and kinase inhibitors revealed that the effects of chronic nicotine treatment are mediated by nicotinic receptors and their downstream effectors including phosphatidylinositol 3-kinase. In contrast to chronic donepezil treatment that enhanced the level of nicotine-induced Ca(2+) influx, chronic nicotine treatment did not significantly alter the level of Ca(2+) influx.
Acetylcholine (ACh) is known to be a key neurotransmitter in the central and peripheral nervous systems, but it is also produced in a variety of non-neuronal tissues and cells, including lymphocytes, placenta, amniotic membrane, vascular endothelial cells, keratinocytes, and epithelial cells in the digestive and respiratory tracts. To investigate contribution made by the high-affinity choline transporter (CHT1) to ACh synthesis in both cholinergic neurons and nonneuronal cells, we transfected rat CHT1 cDNA into NIH3T3ChAT cells, a mouse fibroblast line expressing mouse choline acetyltransferase (ChAT), to establish the NIH3T3ChAT 112-1 cell line, which stably expresses both CHT1 and ChAT. NIH3T3ChAT 112-1 cells showed increased binding of the CHT1 inhibitor [(3)H]hemicholinium-3 (HC-3) and greater [(3)H]choline uptake and ACh synthesis than NIH3T3ChAT 103-1 cells, a CHT1-negative control cell line. HC-3 significantly inhibited ACh synthesis in NIH3T3ChAT 112-1 cells but did not affect synthesis in NIH3T3ChAT 103-1 cells. ACh synthesis in NIH3T3ChAT 112-1 cells was also reduced by amiloride, an inhibitor of organic cation transporters (OCTs) involved in low-affinity choline uptake, and by procaine and lidocaine, two local anesthetics that inhibit plasma membrane phospholipid metabolism. These results suggest that CHT1 plays a key role in ACh synthesis in NIH3T3ChAT 112-1 cells and that choline taken up by OCTs or derived from the plasma membrane is also utilized for ACh synthesis in both cholinergic neurons and nonneuronal cholinergic cells, such as lymphocytes.
The aim of this study was to establish kinetic analysis of [5-(11)C-methoxy]donepezil ([(11)C]donepezil), which was developed for the in-vivo visualization of donepezil binding to acetylcholinesterase (AChE) using positron emission tomography (PET). Donepezil is an AChE inhibitor that is widely prescribed to ameliorate the cognitive impairment of patients with dementia. Six healthy subjects took part in a dynamic study involving a 60-min PET scan after intravenous injection of [(11)C]donepezil. The total distribution volume (tDV) of [(11)C]donepezil was quantified by compartmental kinetic analysis and Logan graphical analysis. A one-tissue compartment model (1TCM) and a two-tissue compartment model (2TCM) were applied in the kinetic analysis. Goodness of fit was assessed with chi(2) criterion and Akaike's Information Criterion (AIC). Compared with a 1TCM, goodness of fit was significantly improved by a 2TCM. The tDVs provided by Logan graphical analysis were slightly lower than those provided by a 2TCM. The rank order of the mean tDVs in 10 regions was in line with the AChE activity reported in a previous post-mortem study. Logan graphical analysis generated voxel-wise images of tDV, revealing the overall distribution pattern of AChE in individual brains. Significant correlation was observed between tDVs calculated with and without metabolite correction for plasma time-activity curves, indicating that metabolite correction could be omitted. In conclusion, this method enables quantitative analysis of AChE and direct investigation of the pharmacokinetics of donepezil in the human brain.
Protection of neurons from neuronal damage and cell death in neurodegenerative disease is a major challenge in neuroscience research. Donepezil, galantamine and tacrine are acetylcholinesterase inhibitors used for the treatment of Alzheimer's disease, and were believed to be symptomatic drugs whose therapeutic effects are achieved by slowing the hydrolysis of acetylcholine at synaptic termini. However, recent accumulated evidence strongly suggests that these acetylcholinesterase inhibitors also possess neuroprotective properties whose mechanism is independent of acetylcholinesterase inhibition. We have shown that acetylcholinesterase inhibitors protect neurons from glutamate-induced neurotoxicity in the primary culture of rat cortical neurons. It was also found that acetylcholinesterase inhibitor treatment induces up-regulation of nicotinic receptor expression levels, a property which may also have some bearing on their therapeutic effects. We next showed that alpha4 and alpha7-nicotinic receptors play important roles in acetylcholinesterase inhibitor-induced neuroprotection and nicotinic receptor up-regulation. Our results also demonstrate the important roles of the phosphatidylinositol 3-kinase pathway downstream of nicotinic receptors in protecting neurons from death and up-regulating nicotinic receptors. This review summarizes recent findings on the roles of the nicotinic receptor in acetylcholinesterase inhibitor-induced neuroprotection and nicotinic receptor up-regulation.
Title: Basic and clinical aspects of non-neuronal acetylcholine: expression of an independent, non-neuronal cholinergic system in lymphocytes and its clinical significance in immunotherapy Fujii T, Takada-Takatori Y, Kawashima K Ref: J Pharmacol Sci, 106:186, 2008 : PubMed
Lymphocytes possess all the components required to constitute an independent, non-neuronal cholinergic system. These include acetylcholine (ACh); choline acetyltransferase (ChAT), its synthesizing enzyme; and both muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively). ACh modifies T and B cell function via both mAChR- and nAChR-mediated pathways. Stimulation of lymphocytes with the T cell activator phytohemagglutinin, protein kinase C activator phorbol ester, or cell surface molecules enhances the synthesis and release of ACh and up-regulates ChAT and/or M(5) mAChR gene expression. Furthermore, animal models of immune disorders exhibit abnormal lymphocytic cholinergic activity. The cholesterol-lowering drug simvastatin attenuates the lymphocytic cholinergic activity of T cells by inhibiting LFA-1 signaling in a manner independent of its cholesterol-lowering activity. This suggests that simvastatin exerts its immunosuppressive effects in part by modifying lymphocytic cholinergic activity. Nicotine, an active ingredient of tobacco, ameliorates ulcerative colitis but exacerbates Crohn's disease. Expression of mRNAs encoding the nAChR alpha7 and alpha5 subunits are significantly diminished in peripheral mononuclear leukocytes from smokers, as compared with those from nonsmokers. In addition, long-term exposure of lymphocytes to nicotine reduces intracellular Ca(2+) signaling via alpha7 nAChR-mediated pathways. In fact, studies of humoral antibody production in M(1)/M(5) mAChR-deficient and alpha7 nAChR-deficient animals revealed the role of lymphocytic cholinergic activity in the regulation of immune function. These results provide clues to understanding the mechanisms underlying immune system regulation and could serve as the basis for the development of new immunomodulatory drugs.
Title: Basic and clinical aspects of non-neuronal acetylcholine: overview of non-neuronal cholinergic systems and their biological significance Kawashima K, Fujii T Ref: J Pharmacol Sci, 106:167, 2008 : PubMed
Acetylcholine (ACh) is a phylogenetically ancient molecule involved in cell-to-cell signaling in almost all life-forms on earth. Cholinergic components, including ACh, choline acetyltransferase, acetylcholinesterase, and muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively) have been identified in numerous non-neuronal cells and tissues, including keratinocytes, cancer cells, immune cells, urinary bladder, airway epithelial cells, vascular endothelial cells, and reproductive organs, among many others. Stimulation of the mAChRs and nAChRs elicits cell-specific functional and biochemical effects. These findings support the notion that non-neuronal cholinergic systems are expressed in certain cells and tissues and are involved in the regulation of their function and that cholinergic dysfunction is related to the pathophysiology of certain diseases. They also provide clues for development of drugs with novel mechanisms of action.
alpha7-nicotinic acetylcholine receptors are one of the most abundant subtypes of nicotinic receptors in the brain and have been shown to be involved in the neuroprotective effect of donepezil. Recently, we showed that in primary culture of rat cortical neurons, chronic donepezil treatment (10 muM, 4 days) (1) induces the up-regulation of alpha7-nicotinic receptors, (2) enhances the nicotine-induced increase in [Ca(2+)](i) and (3) enhances the sensitivity to the neuroprotective effect of donepezil. Here we demonstrate the involvement of alpha7-nicotinic receptors in these three effects. Concomitant treatment with nicotinic receptor antagonist inhibited the up-regulation of alpha7-nicotinic receptor, enhancement of the increase in [Ca(2+)](i) induced by nicotine, and enhancement of sensitivity to the neuroprotective effect of donepezil. Next, using inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase signaling pathways, we demonstrate the involvement of these pathways in the up-regulation of alpha7-nicotinic receptors and in making the neurons more sensitive to the neuroprotective effects of donepezil. Concomitant chronic donepezil treatment with inhibitors of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways inhibited nicotinic receptor up-regulation and enhancement of the response to nicotine, and enhanced the sensitivity to donepezil. This study increases understanding of the less-studied mechanism of chronic donepezil treatment-induced nicotinic receptor up-regulation and increased sensitivity to donepezil.
Previously, we showed that in rat cortical neurons, chronic donepezil treatment (10 microM, 4 days) up-regulates nicotinic receptors (nAChR) and makes neurons more sensitive to the neuroprotective effect of donepezil. Here we examined the mechanism of donepezil-induced neuroprotection in neurons chronically treated with donepezil. The mechanism of neuroprotection was examined under different conditions of exposure to glutamate, acute and moderate, that induce cell death associated with necrotic and apoptotic cell death, respectively. Concomitant treatment with antagonists of nAChRs but not muscarinic receptors inhibited donepezil pretreatment-induced neuroprotection against acute glutamate treatment-induced death. Donepezil pretreatment prevented acute glutamate- and ionomycin-induced neurotoxicity, but not S-nitrosocysteine-induced neurotoxicity, suggesting that donepezil protects neurons via nAChR at levels before nitric oxide synthase activation against acute glutamate neurotoxicity. Concomitant treatment with antagonists of nAChR or phosphatidylinositol 3-kinase (PI3K) signaling inhibitors significantly inhibited neuroprotection against moderate glutamate neurotoxicity and decreased the phosphorylation level of Akt. Neuroprotection was also inhibited by treatment with inhibitor of mitogen-activated protein kinase (MAPK) kinase. These results suggest that donepezil protects neurons against moderate glutamate neurotoxicity via nAChR-PI3K-Akt and MAPK signaling pathways. This study provides novel insight into the mechanism of donepezil-induced neuroprotection that involves nAChR up-regulation.
Bombyx mori, the domesticated silkworm, is a major insect model for research, and the first lepidopteran for which draft genome sequences became available in 2004. Two independent data sets from whole-genome shotgun sequencing were merged and assembled together with newly obtained fosmid- and BAC-end sequences. The remarkably improved new assembly is presented here. The 8.5-fold sequence coverage of an estimated 432 Mb genome was assembled into scaffolds with an N50 size of approximately 3.7 Mb; the largest scaffold was 14.5 million base pairs. With help of a high-density SNP linkage map, we anchored 87% of the scaffold sequences to all 28 chromosomes. A particular feature was the high repetitive sequence content estimated to be 43.6% and that consisted mainly of transposable elements. We predicted 14,623 gene models based on a GLEAN-based algorithm, a more accurate prediction than the previous gene models for this species. Over three thousand silkworm genes have no homologs in other insect or vertebrate genomes. Some insights into gene evolution and into characteristic biological processes are presented here and in other papers in this issue. The massive silk production correlates with the existence of specific tRNA clusters, and of several sericin genes assembled in a cluster. The silkworm's adaptation to feeding on mulberry leaves, which contain toxic alkaloids, is likely linked to the presence of new-type sucrase genes, apparently acquired from bacteria. The silkworm genome also revealed the cascade of genes involved in the juvenile hormone biosynthesis pathway, and a large number of cuticular protein genes.
Title: Roles played by lymphocyte function-associated antigen-1 in the regulation of lymphocytic cholinergic activity Fujii T, Takada-Takatori Y, Kawashima K Ref: Life Sciences, 80:2320, 2007 : PubMed
Lymphocytes possess the essential components of a cholinergic system, including acetylcholine (ACh); choline acetyltransferase (ChAT), its synthesizing enzyme; and both muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively). Stimulation of lymphocytes with phytohemagglutinin, which activates T cells via the T cell receptor/CD3 complex, enhances the synthesis and release of ACh and up-regulates expression of ChAT and M(5) mAChR mRNAs. In addition, activation of protein kinase C and increases in intracellular cAMP also enhance cholinergic activity in T cells, and lymphocyte function associated antigen-1 (LFA-1; CD11a/CD18) is an important mediator of leukocyte migration and T cell activation. Anti-CD11a monoclonal antibody (mAb) as well as antithymocyte globulin containing antibodies against CD2, CD7 and CD11a all increase ChAT activity, ACh synthesis and release, and expression of ChAT and M(5) mAChR mRNAs in T cells. The cholesterol-lowering drug simvastatin inhibits LFA-1 signaling by binding to an allosteric site on CD11a (LFA-1 alpha chain), which leads to immunomodulation. We found that simvastatin abolishes anti-CD11a mAb-induced increases in lymphocytic cholinergic activity in a manner independent of its cholesterol-lowering activity. Collectively then, these results indicate that LFA-1 contributes to the regulation of lymphocytic cholinergic activity via CD11a-mediated pathways and suggest that simvastatin exerts its immunosuppressive effects in part via modification of lymphocytic cholinergic activity.
Immunological activation of T cells enhances synthesis of acetylcholine (ACh) and transcription of choline acetyltransferase (ChAT), M5 muscarinic ACh receptor (mAChR) and acetylcholinesterase (AChE). Stimulation of mAChRs on T and B cells causes oscillating Ca(2+)-signaling and up-regulation of c-fos expression; moreover, M1 mAChRs play a crucial role in the differentiation of CD8(+) T cells into cytolytic T lymphocytes. Collectively, these findings suggest that immune cell function is regulated by its own cholinergic system. Bearing that in mind, we tested whether immune function can be regulated via mAChR-mediated pathways by immunizing combined M1 and M5 mAChR knockout (M1/M5 KO) and wild-type (WT) C57BL/6JJcl mice with ovalbumin (OVA) and measuring serum IgG1 and IgM 1 wk later. We found that serum levels of total and anti-OVA-specific IgG1 were significantly lower in M1/M5 KO than WT mice, though there was no difference in serum levels of total and anti-OVA-specific IgM between the two genotypes. Secretion of interleukin (IL)-6 from activated spleen cells was significantly reduced in M1/M5 KO mice, whereas there was no significant change in gamma interferon secretion. Expression of AChE mRNA was significantly reduced in activated spleen cells from M1/M5 KO mice. These results suggest that M1 and/or M5 mAChRs are involved in regulating cytokine (e.g., IL-6) production, leading to modulation of antibody class switching from IgM to IgG1, but are not involved in the initial generation of the antibody response. They also support the notion that a non-neuronal cholinergic system is involved in regulating immune cell function.
Using a radioimmunoassay (RIA) with high specificity and sensitivity (1 pg/tube) for acetylcholine (ACh), we have been able to measure the ACh content in samples from the bacteria, archaea and eucarya domains of the universal phylogenetic tree. We found detectable levels of ACh to be ubiquitous in bacteria (e.g., Bacillus subtilis), archaea (e.g., Thermococcus kodakaraensis KOD1), fungi (e.g., shiitake mushroom and yeast), plants (e.g., bamboo shoot and fern) and animals (e.g., bloodworm and lugworm). The levels varied considerably, however, with the highest ACh content detected in the top portion of bamboo shoot (2.9 micromol/g), which contained about 80 times that found in rat brain. In addition, using the method of Fonnum, various levels of ACh-synthesizing activity also were detected, a fraction of which was catalyzed by a choline acetyltransferase (ChAT)-like enzyme (sensitive to bromoACh, a selective ChAT inhibitor) in T. kodakaraensis KOD1 (15%), bamboo shoot (91%) and shiitake mushroom (51%), bloodworm (91%) and lugworm (81%). Taken together, these findings demonstrate the ubiquitous expression of ACh and ACh-synthesizing activity among life forms without nervous systems, and support the notion that ACh has been expressed and may be active as a local mediator and modulator of physiological functions since the early beginning of life.
PURPOSE: Cap43 is known as a nickel- and calcium-inducible gene. In the present study, we examined whether 17beta-estradiol (E2) could affect the expression of Cap43 in breast cancer. EXPERIMENTAL DESIGN: Real-time PCR, immunoblotting, and immunocytochemistry were used to examine the expression of Cap43 and estrogen receptor-alpha (ER-alpha) in breast cancer cell lines. MDA-MB-231 and SK-BR-3 cell lines were transfected with ER-alpha cDNA to establish cells overexpressing ER-alpha. Immunohistochemistry was used to evaluate the expression of the Cap43 protein in breast cancer patients (n = 96), and the relationship between Cap43 expression and clinicopathologic findings was examined. RESULTS: Of the eight cell lines, four expressed higher levels of Cap43 with very low levels of ER-alpha, whereas the other four expressed lower levels of Cap43 with high ER-alpha levels. Treatment with E2 decreased the expression of Cap43 dose-dependently in ER-alpha-positive cell lines but not in ER-alpha-negative lines. Administration of antiestrogens, tamoxifen and ICI 182780, abrogated the E2-induced down-regulation of Cap43. Overexpression of ER-alpha in both ER-alpha-negative cell lines, SK-BR-3 and MDA-MB-231, resulted in down-regulation of Cap43. Immunostaining studies showed a significant correlation between Cap43 expression and the histologic grade of tumors (P = 0.0387). Furthermore, Cap43 expression was inversely correlated with the expression of ER-alpha (P = 0.0374). CONCLUSIONS: E2-induced down-regulation of Cap43 seems to be mediated through ER-alpha-dependent pathways in breast cancer cells both in culture and in patients. Cap43 has potential as a molecular marker to determine the therapeutic efficacy of antiestrogenic anticancer agents in breast cancer.
Title: Simvastatin regulates non-neuronal cholinergic activity in T lymphocytes via CD11a-mediated pathways Fujii T, Masuyama K, Kawashima K Ref: Journal of Neuroimmunology, 179:101, 2006 : PubMed
Lymphocyte function associated antigen-1 (LFA-1; CD11a/CD18) is an important mediator of leukocyte migration and T cell activation. We previously showed that antithymocyte globulin stimulates an independent, non-neuronal cholinergic system in T cells via LFA-1-mediated pathways, as evidenced by increases in acetylcholine (ACh) synthesis and choline acetyltransferase (ChAT) mRNA expression. The cholesterol-lowering drug simvastatin inhibits LFA-1 signaling by binding to an allosteric site on CD11a (LFA-1 alpha chain), which leads to immunomodulation. In the present study, we investigated whether simvastatin modulates lymphocytic cholinergic activity in T cells. We found that anti-CD11a monoclonal antibody (mAb) increased ChAT activity, ACh synthesis and release, and expression of ChAT and M5 muscarinic ACh receptor mRNA in MOLT-3 cells, a human leukemic T cell line. Simvastatin abolished these anti-CD11a mAb-induced increases in lymphocytic cholinergic activity in a manner independent of its cholesterol-lowering activity. These results indicate that LFA-1 contributes to the regulation of lymphocytic cholinergic activity via CD11a-mediated pathways, and suggest that simvastatin exerts its immunosuppressive effects in part via modification of lymphocytic cholinergic activity.
Gordonia sp. strain P8219, a strain able to decompose di-2-ethylhexyl phthalate, was isolated from machine oil-contaminated soil. Mono-2-ethylhexyl phthalate hydrolase was purified from cell extracts of this strain. This enzyme was a 32,164-Da homodimeric protein, and it effectively hydrolyzed monophthalate esters, such as monoethyl, monobutyl, monohexyl, and mono-2-ethylhexyl phthalate. The K(m) and V(max) values for mono-2-ethylhexyl phthalate were 26.9 +/- 4.3 microM and 18.1 +/- 0.9 micromol/min . mg protein, respectively. The deduced amino acid sequence of the enzyme exhibited less than 30% homology with those of meta-cleavage hydrolases which are serine hydrolases but exhibited no significant homology with the sequences of serine esterases. The pentapeptide motif GXSXG, which is conserved in serine hydrolases, was present in the sequence. The enzymatic properties and features of the primary structure suggested that this enzyme is a novel enzyme belonging to an independent group of serine hydrolases.
We examined the mechanisms of the neuroprotective effects of two central-type acetylcholinesterase inhibitors, galanthamine and tacrine, on nitric oxide-mediated glutamate neurotoxicity using primary cultures from the cerebral cortex of fetal rats. Galanthamine and tacrine showed prominent protective effects against glutamate neurotoxicity. Mecamylamine, a nicotinic acetylcholine receptor antagonist, but not scopolamine, a muscarinic acetylcholine receptor antagonist, inhibited the protective effects of these inhibitors on glutamate neurotoxicity. Furthermore, dihydro-beta-erythroidine, an alpha4-nicotinic receptor antagonist, and methyllycaconitine, an alpha7-nicotinic receptor antagonist, inhibited the neuroprotective effects of galanthamine but not tacrine. Next, we investigated the site of action where galanthamine and tacrine prevent glutamate neurotoxicity. Both these acetylcholinesterase inhibitors prevented glutamate- and ionomycin-induced neurotoxicity, but only tacrine prevented S-nitrosocysteine-induced neurotoxicity. These results suggest that galanthamine and tacrine protect cortical neurons from glutamate neurotoxicity via different mechanisms.
Title: Rational control of enantioselectivity of lipase by site-directed mutagenesis based on the mechanism Ema T, Fujii T, Ozaki M, Korenaga T, Sakai T Ref: Chem Commun (Camb), :4650, 2005 : PubMed
The enantioselectivity of a Burkholderia cepacia lipase toward secondary alcohols could be both increased and decreased rationally by introducing only a single mutation on the basis of the mechanism proposed previously.
Title: Structure of the carboxypeptidase Y inhibitor IC in complex with the cognate proteinase reveals a novel mode of the proteinase-protein inhibitor interaction Mima J, Hayashida M, Fujii T, Narita Y, Hayashi R, Ueda M, Hata Y Ref: Journal of Molecular Biology, 346:1323, 2005 : PubMed
Carboxypeptidase Y (CPY) inhibitor, IC, shows no homology to any other known proteinase inhibitors and rather belongs to the phosphatidylethanolamine-binding protein (PEBP) family. We report here on the crystal structure of the IC-CPY complex at 2.7 A resolution. The structure of IC in the complex with CPY consists of one major beta-type domain and a N-terminal helical segment. The structure of the complex contains two binding sites of IC toward CPY, the N-terminal inhibitory reactive site (the primary CPY-binding site) and the secondary CPY-binding site, which interact with the S1 substrate-binding site of CPY and the hydrophobic surface flanked by the active site of the enzyme, respectively. It was also revealed that IC had the ligand-binding site, which is conserved among PEBPs and the putative binding site of the polar head group of phospholipid. The complex structure and analyses of IC mutants for inhibitory activity and the binding to CPY demonstrate that the N-terminal inhibitory reactive site is essential both for inhibitory function and the complex formation with CPY and that the binding of IC to CPY constitutes a novel mode of the proteinase-protein inhibitor interaction. The unique binding mode of IC toward the cognate proteinase provides insights into the inhibitory mechanism of PEBPs toward serine proteinases and into the specific biological functions of IC belonging to the PEBP family as well.
Acetylcholine (ACh) is known generally as the neurotransmitter in the mammalian central and peripheral cholinergic nervous systems. However, ACh is also widely expressed in non-neuronal animal tissues and in plants, fungi and bacteria, where it is likely involved in the transport of water, electrolytes and nutrients, and in modulating various other cell functions. We have investigated the expression of ACh and ACh-synthesizing activity in various strains of Archaea, which are situated between Bacteria and Eucarya in the universal phylogenetic tree. Using a sensitive and specific radioimmunoassay, differing levels of ACh were detected in the Hyperthermophiles Thermococcus kodakaraensis KOD1, Sulfolobus tokodaii strain 7 and Pyrobaculum calidifontis VA1; the Methanogens Methanothermobacter thermautotrophicus deltaH and Methanosarcina barkeri; and the Halophiles Halobacterium sp. NRC-1 and Haloferax volcanii. T. kodakaraensis KOD1 expressed the highest levels of ACh among the Archaea tested; moreover, the substance expressed was verified to be ACh using high-performance liquid chromatography with electrochemical detection. Varying degrees of ACh-synthesizing activity were also identified in all of the strains, and the activity of bromoACh-sensitive choline acetyltransferase, an enzyme responsible for ACh synthesis in the nervous system, was detected in T. kodakaraensis KOD1. Our findings demonstrate that ACh and ACh-synthesizing activity are both expressed in evolutionally old Archaea. In the context of the recent discovery of non-neuronal ACh in bacteria, fungi, plants and animals, these findings support the notion that ACh has been expressed in organisms from the origin of life on the earth, functioning as a local mediator as well as a neurotransmitter.
Title: [An independent, non-neuronal cholinergic system in lymphocytes and its roles in regulation of immune function] Fujii T Ref: Nihon Yakurigaku Zasshi, 123:179, 2004 : PubMed
Acetylcholine (ACh) is classically thought of as a neurotransmitter in mammalian species. However, lymphocytes express most of the cholinergic components found in the nervous system, including ACh, choline acetyltransferase (ChAT), high-affinity choline transporter, and acetylcholinesterase as well as both muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively). Activation of T cells via the T cell receptor/CD3 complex, contact of T cells with antigen presenting cells, or activation of the adenylyl cyclase pathway in T cells modulates cholinergic activity, as evidenced by up-regulation of ChAT and M(5) mAChR mRNA expression. Stimulation of mAChRs on T and B cells with ACh or another mAChR agonists elicits intracellular Ca(2+) signaling, up-regulation of c-fos expression, increased nitric oxide synthesis and interleukin-2-induced signal transduction via M(3) and M(5) mAChR-mediated pathways. Acute stimulation of nAChRs with ACh or nicotine causes rapid and transient Ca(2+) signaling in T and B cells, probably via alpha7 nAChRs subunit-mediated pathways. Chronic nicotine stimulation, by contrast, down-regulates nAChR expression and suppresses T cell activity. Abnormalities in lymphocytic cholinergic system have been seen in animal models of immune deficiency and immune acceleration. Collectively, these data provided a compelling picture in which immune function is, at least partly, under the control of an independent, non-neuronal cholinergic system in lymphocytes.
Title: Expression of non-neuronal acetylcholine in lymphocytes and its contribution to the regulation of immune function Kawashima K, Fujii T Ref: Front Biosci, 9:2063, 2004 : PubMed
Lymphocytes express most components of the cholinergic system including acetylcholine (ACh), muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively), choline acetyltransferase (ChAT), high affinity choline transporter and acetylcholinesterase. ACh and mAChR agonists elicit intracellular Ca2+ signaling, up-regulation of c-fos expression and nitric oxide synthesis within T and B cells probably via M3 and M5 mAChRs. Stimulation of nAChRs with ACh or nicotine causes a rapid and transient Ca2+ signaling in T and B cells, probably via alpha7 nAChR subunit-mediated pathways. Phytohemagglutinin- or antigen-induced T cell activation via cell surface molecules (e.g., T cell receptor/CD3 complexes) enhances lymphocytic cholinergic transmission by up-regulating ChAT and M5 mAChR expression. It is thus likely that a local lymphocytic cholinergic system is involved in regulating immune function. This idea is supported by the findings that lymphocytic cholinergic activity is altered in animal models exhibiting immunological abnormalities. In addition, it appears likely that during interactions mediated by cell surface molecules T cells communicate via ACh with thymic epithelial cells and vascular endothelial cells, which also express ChAT and nAChRs or mAChRs. This interaction leads to T cell selection and maturation in the thymus and local vascular smooth muscle relaxation. Collectively, these data provide a compelling picture in which lymphocytes constitute a cholinergic system that is independent of cholinergic nerves, and which is involved in the regulation of immune function and local circulation.
Title: Crystallization and preliminary X-ray analysis of carboxypeptidase Y inhibitor IC complexed with the cognate proteinase Mima J, Hayashida M, Fujii T, Hata Y, Hayashi R, Ueda M Ref: Acta Crystallographica D Biol Crystallogr, 60:1622, 2004 : PubMed
Carboxypeptidase Y (CPY) inhibitor I(C) is a naturally occurring serine carboxypeptidase inhibitor from Saccharomyces cerevisiae, the sequence of which is not homologous with any other known proteinase inhibitor and is classified as the phosphatidylethanolamine-binding protein (PEBP). I(C) has been crystallized in complex with the deglycosylated form of CPY by the hanging-drop vapour-diffusion technique with ammonium sulfate as a precipitant. The crystals of the complex belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 81.13, b = 186.6, c = 65.14 A. Diffraction data were collected to 2.7 A resolution. Structure determination of the complex is in progress by the molecular-replacement method using the structure of CPY as a search model.
We used a selective EP4 receptor agonist, ONO-4819, and a human leukemic T cell line MOLT-3 cells, which express all four prostaglandin E2 (PGE2) receptors (EP1-EP4), to investigate whether the EP4 PGE2 receptor subtype is involved in regulating lymphocytic cholinergic activity. Phytohemagglutinin (PHA), a T cell activator, significantly enhanced the expression of EP4 receptor mRNA during the first 3-6 h of exposure, after which, expression gradually declined. Furthermore, PHA stimulation slightly but significantly up-regulated the expression of EP2 mRNA after 12 h and of EP3 mRNA after 6 h. By contrast, expression level of EP1 receptor mRNA was not affected by PHA. ONO-4819 (1 microM), which was added to cultures after 3 h of PHA stimulation, significantly increased cellular ACh content and release, and up-regulated ChAT mRNA expression and activity but inhibited MOLT-3 cell proliferation. These findings suggest that the activation of T lymphocytes up-regulates EP4 receptor mRNA expression and, to a lesser extent, EP2 and EP3 receptors and that PGE2 enhances nonneuronal lymphocytic cholinergic transmission in activated T cells, at least in part, via EP4 receptor-mediated pathways.
Title: Detection of the high-affinity choline transporter in the MOLT-3 human leukemic T-cell line Fujii T, Okuda T, Haga T, Kawashima K Ref: Life Sciences, 72:2131, 2003 : PubMed
We previously showed that lymphocytes possess the necessary components to constitute an independent, non-neuronal cholinergic system; these include acetylcholine (ACh) itself, choline acetyltransferase (the ACh-synthesizing enzyme), and both muscarinic and nicotinic ACh receptors (AChRs). In addition, we showed that stimulation of AChRs with their respective agonists elicits a variety of biochemical and functional effects, suggesting that lymphocytic cholinergic system is involved in the regulation of immune function. In nerve terminals, choline taken up via the high-affinity choline transporter (CHT1) is exclusively utilized for ACh synthesis. In the present study, therefore, we investigated the expression of CHT1 in T-lymphocytes. Reverse transcription-polymerase chain reaction analysis revealed that MOLT-3 cells, a human leukemic T-cell line used as a T-lymphocyte model, expressed CHT1 mRNA, but that the CEM and Jurkat T-cell lines did not. Consistent with that finding, specific binding of [3H]hemicholinium-3 (HC-3), an inhibitor of CHT1, and HC-3-sensitive [3H]choline uptake were also detected in MOLT-3 cells. These results suggest that CHT1 plays a role in mediating choline uptake in T-lymphocytes and provides further evidence for the presence of an independent lymphocytic cholinergic system.
Title: Upregulation of mRNA encoding the M5 muscarinic acetylcholine receptor in human T- and B-lymphocytes during immunological responses Fujii T, Watanabe Y, Inoue T, Kawashima K Ref: Neurochem Res, 28:423, 2003 : PubMed
Lymphocytes possess an independent, non-neuronal cholinergic system. Moreover, both T- and B-lymphocytes express multiple muscarinic acetylcholine receptors (mAChR). To obtain a better understanding of the regulatory mechanisms governing mAChR gene expression in the lymphocytic cholinergic system, we examined the effects of lymphocyte activation on expression of mAChR mRNA. Stimulation of T- and B-lymphocytes, respectively, with T-cell activator phytohemagglutinin and B-cell activator Staphylococcus aureus Cowan I upregulated M5 mAChR mRNA expression in the CEM human leukemic T-cell line and in the Daudi B-cell line, which served as models of lymphocytes. In striking contrast, M3 and M4 mAChR mRNA expression was not affected in either cell line. Nonetheless, stimulating lymphocytes with phorbol 12-myristate 13-acetate, a protein kinase C activator, plus ionomycin, a calcium ionophore, upregulated expression of both M3 and M5 mAChR mRNA. This represents the first demonstration that immunological stimulation leads to M5 mAChR gene expression in lymphocytes.
Title: Acetylcholine increase in amniotic fluid of experimental rats for intrauterine growth retardation Horikoshi T, Fujii T, Kawashima K, Sakuragawa N Ref: Life Sciences, 72:2145, 2003 : PubMed
Previous reports from this laboratory have demonstrated evidence for synthesis and release of acetylcholine (ACh) and catecholamines (CAs) by human amniotic epithelial cells (HAEC) and the presence of ACh and CAs in amniotic fluid. To study the physiological role of amniotic ACh, we used an experimental pregnant rat model for intrauterine growth retardation. Prior to this experiment, we confirmed the presence of choline acetyltransferase in the HAEC by immunocytochemical staining. Amniotic fluid was collected at 48 and 72 h after a transient ligation of the uterine vessels near the lower and upper ends of the right horn of the pregnant rat. The ACh concentration in the amniotic fluid from rats received intrauterine ischemia increased with time to a greater degree compared with the control rat, although the increase was not statistically significant. These results suggest that intrauterine hypoxic conditions cause a tendency to increase ACh concentrations in the amniotic fluid.
Title: The endogenous, immunologically active peptide apelin inhibits lymphocytic cholinergic activity during immunological responses Horiuchi Y, Fujii T, Kamimura Y, Kawashima K Ref: Journal of Neuroimmunology, 144:46, 2003 : PubMed
We investigated the effects of apelin, an immunologically active peptide ligand for orphan receptor APJ, on acetylcholine (ACh) synthesis in MOLT-3 human leukemic T cells. We initially confirmed expression of APJ mRNA in several human T- and B-cell lines by reverse transcription-polymerase chain reaction (RT-PCR). We also found that in phytohemagglutinin (PHA)-stimulated MOLT-3 cells, an active apelin fragment, apelin-13, down-regulates expression of choline acetyltransferase (ChAT) mRNA and significantly reduces ChAT activity and cellular ACh content and release. It thus appears that apelin inhibits lymphocytic cholinergic activity via APJ during immunological responses.
Acetylcholine (ACh) is a well-known neurotransmitter in the cholinergic nervous systems of vertebrates and insects; however, there is only indirect evidence for its presence in lower invertebrates, such as plants and fungi. We therefore investigated the expression of ACh in invertebrates (sea squirt, sea urchin, trepang, squid, abalone, nereis, sea anemone, coral and sponge), plants (arabidopsis, eggplant, bamboo shoot, cedar, hinoki, pine, podcarp, fern, horsetail and moss), fungi (yeast and mushroom) and bacteria by assaying ACh content and synthesis, focusing on the presence of two synthetic enzymes, choline acetyltransferase (ChAT) and carnitine acetyltransferase (CarAT). Using a specific radioimmunoassay, ACh was detected in all samples tested. The levels varied considerably, however, with the upper portion of bamboo shoots having the highest content (2.9 micromol/g). ACh synthesis was also detected in all samples tested; moreover, the activity in most samples from the animal kingdom, as well as bamboo shoots and the stem of the shiitake mushroom, were sensitive to both ChAT and CarAT inhibitors. Levels of ACh synthesis were lower in samples from other plants, fungi and bacteria and were insensitive to ChAT and CarAT inhibitors. These findings demonstrate the presence of ACh and ACh-synthesizing activity in evolutionally primitive life as well as in more complex multicellular organisms. In the context of the recent discovery of non-neuronal ACh in various mammalian species, these findings suggest that ACh been expressed in organisms from the beginning of life, functioning as a local mediator as well as a neurotransmitter.
Title: Nitric oxide (NO) synthase mRNA expression and NO production via muscarinic acetylcholine receptor-mediated pathways in the CEM, human leukemic T-cell line Kamimura Y, Fujii T, Kojima H, Nagano T, Kawashima K Ref: Life Sciences, 72:2151, 2003 : PubMed
Nitric oxide (NO) is synthesized from L-arginine by neuronal, endothelial and inducible isoforms of NO synthase (nNOS, eNOS and iNOS, respectively) and is involved in the regulation of a variety of physiological functions, including immune activity. In vascular endothelial cells, stimulation of M(3) subtype of muscarinic acetylcholine receptors (mAChRs) triggers NO synthesis by eNOS. Human lymphocytes express several mAChR subtypes and their stimulation increases the intracellular free Ca(2+) concentration and up-regulates c-fos gene expression. While the above findings suggest involvement of the lymphocytic cholinergic system in the regulation of immune function, little is known on NOS expression and NO synthesis in T-lymphocytes. In the present study, using reverse transcription-polymerase chain reaction, we found that CEM cells express mRNAs encoding iNOS and nNOS, but not for eNOS. In addition, using quantitative fluorescence microscopy and a novel NO-sensitive fluorescent indicator, DAF-2, we found that oxotremorine-M (Oxo-M) (100 microM), a non-selective mAChR agonist, enhances NO production in the cells. This effect of Oxo-M was antagonized by pirenzepine (10 microM), an antagonist acting preferentially at M(1) mAChR and by atropine (10 microM). Also 4-DAMP (10 microM), an antagonist acting preferentially at M(3) mAChR, reduced significantly the effect of Oxo-M, while AFDX-116 (10 microM), an antagonist acting preferentially at M(2) mAChR, was ineffective. These findings suggest that T-lymphocytes express functional mAChRs linked to NO synthesis by nNOS and/or iNOS.
Title: The lymphocytic cholinergic system and its contribution to the regulation of immune activity Kawashima K, Fujii T Ref: Life Sciences, 74:675, 2003 : PubMed
Lymphocytes express most of the cholinergic components found in the nervous system, including acetylcholine (ACh), choline acetyltransferase (ChAT), high affinity choline transporter, muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively), and acetylcholinesterase. Stimulation of T and B cells with ACh or another mAChR agonist elicits intracellular Ca2+ signaling, up-regulation of c-fos expression, increased nitric oxide synthesis and IL-2-induced signal transduction, probably via M3 and M5 mAChR-mediated pathways. Acute stimulation of nAChRs with ACh or nicotine causes rapid and transient Ca2+ signaling in T and B cells, probably via alpha7 nAChR subunit-mediated pathways. Chronic nicotine stimulation, by contrast, down-regulates nAChR expression and suppresses T cell activity. Activation of T cells with phytohemagglutinin or antibodies against cell surface molecules enhances lymphocytic cholinergic transmission by activating expression of ChAT and M5 mAChR, which is suggestive of local cholinergic regulation of immune system activity. This idea is supported by the facts that lymphocytic cholinergic activity reflects well the changes in immune system function seen in animal models of immune deficiency and immune acceleration. Collectively, these data provide a compelling picture in which lymphocytes constitute a cholinergic system that is independent of cholinergic nerves, and which is involved in the regulation of immune function.
Title: The lymphocytic cholinergic system and its biological function Kawashima K, Fujii T Ref: Life Sciences, 72:2101, 2003 : PubMed
Lymphocytes are now known to possess the essential components for a non-neuronal cholinergic system. These include acetylcholine (ACh); choline acetyltransferase (ChAT), its synthesizing enzyme; and both muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively). Stimulating lymphocytes with phytohemagglutinin, a T-cell activator; Staphylococcus aureus Cowan I, a B-cell activator; or cell surface molecules enhances the synthesis and release of ACh and up-regulates expression of ChAT and M(5) mAChR mRNAs. Activation of mAChRs and nAChRs on lymphocytes elicits increases in the intracellular Ca(2+) concentration and stimulates c-fos gene expression and nitric oxide synthesis. On the other hand, long-term exposure to nicotine down-regulates expression of nAChR mRNA. Abnormalities in the lymphocytic cholinergic system have been detected in spontaneously hypertensive rats and MRL-lpr mice, two animal models of immune disorders. Taken together, these data present a compelling picture in which immune function is, at least in part, under the control of an independent non-neuronal lymphocytic cholinergic system.
Title: Nicotine-induced Ca2+ signaling and down-regulation of nicotinic acetylcholine receptor subunit expression in the CEM human leukemic T-cell line Kimura R, Ushiyama N, Fujii T, Kawashima K Ref: Life Sciences, 72:2155, 2003 : PubMed
We previously showed that T- and B-lymphocytes express both muscarinic and nicotinic acetylcholine (ACh) receptors (mAChR and nAChR, respectively), and that stimulation of M(3) mAChRs on lymphocytes increases the intracellular free Ca(2+) concentration ([Ca(2+)](i)) and up-regulates c-fos gene expression. Little is known about the effects of nicotinic stimulation on lymphocyte function, however. We therefore investigated the acute effect of nicotine on [Ca(2+)](i) in CEM cells, a model of T-lymphocytes, using confocal laser scanning microscopy with fluo-3, a Ca(2+)-sensitive fluorescent indicator. In addition, we examined the long-term effect of nicotine on the expression of selected nAChR subunits using semiquantitative reverse transcription-polymerase chain reaction analysis. In the presence of extracellular Ca(2+), nicotine (30 microM) evoked rapid, transient increases in [Ca(2+)](i). This effect was concentration-dependently inhibited by the alpha7 nAChR subunit antagonists, alpha-bungarotoxin (0.01-10 microM) and methyllycaconitine (0.01-10 mM), suggesting that the alpha7 nAChR subunit mediates Ca(2+) signaling in T-lymphocytes. Nicotine (0.01-10 microM) also concentration-dependently down-regulated expression of mRNAs for all the nAChR subunits tested: expression of the alpha6 and alpha7 subunits was down-regulated within 1 week, while expression of the alpha3 and alpha5 subunits declined gradually throughout the 8-week experimental period. These findings indicate that nicotine--and therefore likely smoking--affects immune function by suppressing expression of the neuronal nAChR subtype involved in Ca(2+) signaling in lymphocytes.
Title: Expression of multiple mRNA species for choline acetyltransferase in human T-lymphocytes Ogawa H, Fujii T, Watanabe Y, Kawashima K Ref: Life Sciences, 72:2127, 2003 : PubMed
Acetylcholine (ACh) is synthesized by choline acetyltransferase (ChAT) in cholinergic neurons. However, both ACh and mRNA for ChAT are expressed in mononuclear leukocytes and various human leukemic T-cell lines. Multiple ChAT mRNA species (R-, N0-, N1-, N2-, and M-types) having an identical coding region and different 5'-noncoding regions have been discovered in human brain and spinal cord. These mRNAs are transcribed by a combination of use of different promoter regions and alternative splicing. However, which types of ChAT mRNA species are expressed in T-lymphocytes remains to be elucidated. In the present study, we used two human leukemic T-cell lines, CCRF-CEM (CEM) and MOLT-3, which express the same ChAT mRNA as that in the nervous system. Major mRNA species in CEM were N2- and M-type, and to a lesser extent N1-type, while MOLT-3 expressed only N2-type. Neither CEM nor MOLT-3 expressed R-type mRNA. We previously found a lack of mRNA expression encoding vesicular acetylcholine transporter (VAChT) in CEM and MOLT-3, which mediates ACh transport to synaptic vesicles in cholinergic neurons. These findings suggest that the mechanisms regulating ChAT mRNA expression in T-lymphocytes differ from those in cholinergic neurons.
Title: Effects of human antithymocyte globulin on acetylcholine synthesis, its release and choline acetyltransferase transcription in a human leukemic T-cell line Fujii T, Ushiyama N, Hosonuma K, Suenaga A, Kawashima K Ref: Journal of Neuroimmunology, 128:1, 2002 : PubMed
Lymphocytes possess an independent, nonneuronal cholinergic system. In the present study, we investigated the short- and long-term effects of antithymocyte globulin (ATG)-Fresenius (ATG-F), a human antithymocyte globulin that binds to CD2, CD7 and CD11a, on acetylcholine (ACh) synthesis and transcription of choline acetyltransferase (ChAT) in CCRF-CEM cells, a human leukemic T-cell line. In the short-term (6 h), ATG-F enhanced ACh release, likely through transient increases in intracellular Ca(2+) ([Ca(2+)](i)) mediated by CD7, which led to declines in intracellular ACh content. By 48 h, however, the ACh content had increased as compared to control due to up-regulation of ChAT expression mediated by CD11a.
Title: An independent non-neuronal cholinergic system in lymphocytes Fujii T, Kawashima K Ref: Japanese Journal of Pharmacology, 85:11, 2001 : PubMed
Acetylcholine (ACh) is a well characterized neurotransmitter occurring throughout the animal kingdom. In addition, both muscarinic and nicotinic ACh receptors have been identified on lymphocytes of various origin, and their stimulation by muscarinic or nicotinic agonists elicits a variety of functional and biochemical effects. It was thus initially postulated that the parasympathetic nervous system may play a role in modulating immune system function. However, ACh in the blood has now been localized to lymphocytes; indeed expression of choline acetyltransferase (ChAT), an ACh synthesizing enzyme, has been shown in human blood mononuclear leukocytes, human leukemic T-cell lines and rat lymphocytes. Stimulation of T-lymphocytes with phytohemagglutinin activates the lymphoid cholinergic system, as evidenced by increased synthesis and release of ACh and increased expression of mRNAs encoding ChAT and ACh receptors. The observation that M3 muscarinic receptor stimulation by ACh and other agonists increases the intracellular free Ca2+ concentration and upregulates c-fos gene expression strongly argues that ACh, synthesized and released from T-lymphocytes, acts as an autocrine and/or paracrine factor regulating immune function. These findings present a compelling picture in which immune function is, at least in part, under the control of an independent lymphoid cholinergic system.
Title: Decreased acetylcholine content and choline acetyltransferase mRNA expression in circulating mononuclear leukocytes and lymphoid organs of the spontaneously hypertensive rat Fujimoto K, Matsui M, Fujii T, Kawashima K Ref: Life Sciences, 69:1629, 2001 : PubMed
It has been confirmed that the neurotransmitter acetylcholine (ACh) is present in blood; it is synthesized in T-lymphocytes by choline acetyltransferase (ChAT) and released upon T-lymphocyte activation. Both muscarinic and nicotinic ACh receptors have been identified on lymphocytes isolated from thymus, lymph node, spleen and blood, and their stimulation by muscarinic and nicotinic agonists elicits a variety of functional and biochemical effects, providing a strong argument that ACh synthesized and released from T-lymphocytes acts as an autocrine and/or paracrine factor regulating immune function. In the present study, we compared ACh levels in the blood, circulating mononuclear leukocytes (MNLs), thymus and spleen of spontaneously hypertensive rats (SHRs), which exhibit immune deficiencies related to the emergence of natural thymocytotoxic autoantibody, age-related decline of T-cell function and morphological changes in immune organs, with ACh levels in age-matched, normotensive Wistar Kyoto rats. In each case, ACh levels in 5-, 10- and 20-week-old SHRs were significantly lower than in WKYs. ChAT mRNA expression in MNLs was also significantly depressed in the SHRs. These results suggest that diminished synthesis and release of ACh from MNLs into blood and lymphoid organs likely reflects an immune deficiency related to T-cell dysfunction.
Title: Non-neuronal neurotransmitters and neurotrophic factors in amniotic epithelial cells: expression and function in humans and monkey Sakuragawa N, Elwan MA, Uchida S, Fujii T, Kawashima K Ref: Japanese Journal of Pharmacology, 85:20, 2001 : PubMed
Human amniotic epithelial cells (HAEC) are formed from epiblasts on the 8th day after fertilization. Because they lack major histocompatibility complex (MHC) antigen, human amniotic tissue transplantation has been used for allotranplantation to treat patients with lysosomal diseases. We have provided evidence that HAEC have multiple functions such as synthesis and release of acetylcholine (ACh) and catecholamine (CA) as well as expressing mRNA coding for dopamine receptors and dopamine (DA) transporter (DAT). On the other hand, we showed that monkey amniotic epithelial cells (MAEC) synthesize and release CA and posses DA receptors and DAT. Detection of muscarinic actylcholine receptors indicates the presence of an autocrine mechanism in HAEC. Recently, we found that HAEC have neurotrophic function in conditioned medium from HAEC, indicating the presence of a novel neurotrohpic factor that is synthesized and released from HAEC. The amniotic membrane may have a significant role in supplying neurotrophic factors as well as neurotransmitters to the amniotic fluid, suggesting an important function in the early stages of neural development of the embryo. This review will focus on the neuropharmacological aspects of HAEC and MAEC in relation to the physiology of amniotic membrane.
Title: Enhancement of hippocampal cholinergic neurotransmission through 5-HT1A receptor-mediated pathways by repeated lithium treatment in rats Fujii T, Nakai K, Nakajima Y, Kawashima K Ref: Canadian Journal of Physiology & Pharmacology, 78:392, 2000 : PubMed
Hippocampal cholinergic neuronal activity is reported to be regulated, at least partly, through serotonin1A (5-HT1A) receptors. Chronic lithium treatment has been shown to alter both behavioral and neurochemical responses mediated by postsynaptic 5-HT1A receptors. We investigated whether long-term lithium treatment affects central cholinergic neurotransmission through 5-HT1A receptor-mediated pathways. Changes in acetylcholine (ACh) release induced by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT1A receptor agonist, in the rat hippocampus were measured using a microdialysis technique and a radioimmunoassay for ACh. Administration of lithium for 21 days resulted in a serum lithium concentration of 1.03 mM and caused little change in density or affinity of [3H]8-OH-DPAT binding sites in the hippocampus. The local application of 8-OH-DPAT into the hippocampus of lithium treated rats increased the ACh efflux in both the absence and the presence of physostigmine, a cholinesterase (ChE) inhibitor, in the perfusion fluid. The basal ACh efflux of lithium treated rats was not different from that of the control rats under normal conditions, but was significantly higher than that of the controls when ChE was inhibited. These results demonstrate that chronic lithium treatment increases spontaneous ACh release in the hippocampus under conditions of ChE inhibition, but not under normal conditions, and enhances cholinergic neurotransmission through 5-HT1A receptor-mediated pathways, and suggest that activation of 5-HT1A receptor function by lithium is related to the enhancement of hippocampal cholinergic neurotransmission.
Title: Effects of physostigmine and calcium on acetylcholine efflux from the hippocampus of freely moving rats as determined by in vivo microdialysis and a radioimmunoassay Fujii T, Harada H, Koyama T, Nakajima Y, Kawashima K Ref: Neuroscience Letters, 289:181, 2000 : PubMed
The effects varying the concentration of Ca2+ in perfused artificial cerebrospinal fluid ([Ca2+]csf) on basal acetylcholine (ACh) efflux from the hippocampus of freely moving rats, in the presence and absence of the cholinesterase (ChE) inhibitor physostigmine, were investigated using in vivo microdialysis and a highly specific radioimmunoassay for ACh. In the absence of physostigmine, basal ACh efflux was 3.4+/-0.7 pg/30 min (mean +/- SEM) at [Ca2+]csf = 1.26 mM. Stepwise increases in [Ca2+]csf elicited a gradual increase in ACh efflux that was significant at [Ca2+]csf = 5.04 mM. Inhibition of ChE by addition of 10 microM physostigmine to the perfusate increased the efflux of ACh to 103.2+/-21.1 pg/30 min ([Ca2+]csf = 1.26 mM), and the efflux was augmented still further by increasing [Ca2+]csf, a change that became significant at [Ca2+]csf = 3.78. These results illustrate the sensitivity of basal ACh efflux from the hippocampus to changes in the extracellular Ca2+ concentration, and suggest that a more accurate picture of hippocampal cholinergic activity is obtained by microdialysis using normal artificial cerebrospinal fluid, under physiological conditions, rather than in the presence of a ChE inhibitor.
Title: Ca2+ oscillation and c-fos gene expression induced via muscarinic acetylcholine receptor in human T- and B-cell lines Fujii T, Kawashima K Ref: Naunyn Schmiedebergs Arch Pharmacol, 362:14, 2000 : PubMed
We previously reported that blood acetylcholine (ACh) mainly originates from T-lymphocytes and that muscarinic (Ms) ACh receptor mRNA is expressed in both T- and B-lymphocytes. In the present study, we used confocal laser scanning microscopy and fluo-3, a calcium-sensitive indicator, to investigate the effects of Ms-ACh receptor agonists on the intracellular free Ca2+ concentration ([Ca2+]i) in single cells from human T-cell (CEM) and B-cell (Daudi) lines, which we used as models of lymphocytes. In both cell lines, stimulation of Ms-ACh receptors with ACh (0.1-100 microM), bethanechol (100 microM), car-bachol (100 microM) or oxotremorine-M (Oxo-M; 0.1-100 microM) induced [Ca2+]i-dependent increases in fluo-3 fluorescence, which in the presence of extracellular Ca2+ were followed by oscillations in [Ca2+]i that persisted for at least 10 min. All effects were completely blocked by atropine (1 microM), an Ms-ACh receptor antagonist. In both cell lines Oxo-M (100 microM) up-regulated expression of c-fos mRNA in an extracellular Ca2+-dependent manner. Again, the effect was blocked by 1 microM atropine. These results provide the first evidence that stimulation of Ms-ACh receptors induces Ca2+ oscillations and up-regulates c-fos gene expression in T- and B-lymphocytes, which is consistent with the notion that ACh released from T-lymphocytes triggers nuclear signaling via Ms-ACh receptors.
Title: Calcium signaling and c-Fos gene expression via M3 muscarinic acetylcholine receptors in human T- and B-cells Fujii T, Kawashima K Ref: Japanese Journal of Pharmacology, 84:124, 2000 : PubMed
We previously showed that blood acetylcholine (ACh) originates mainly from T-lymphocytes, and that stimulation of muscarinic ACh receptors (mAChRs) induces Ca2+ oscillations and up-regulates c-fos gene expression in both T- and B-lymphocytes. In the present study, we investigated which mAChR subtypes are involved in Ca2+ signaling and c-fos gene expression in human T- (CEM) and B- (Daudi) cells. Stimulation of mAChRs with 100 microM oxotremorine-M, an M1/M3 agonist, increased levels of intracellular free Ca2+ ([Ca2+]i) and c-fos mRNA expression in both cell lines. 4-DAMP, an M3 antagonist, more effectively blocked the oxotremorine-M-induced increase in [Ca2+]i than pirenzepine and telenzepine, M1-receptor antagonists; AF-DX 116, an M2 antagonist; hexahydrosiladifenidol, a weak M3 antagonist; or hexamethonium and d-tubocurarine, nicotinic receptor antagonists. McN-A-343 (100 microM), a partial M1-receptor agonist, had no apparent effect on [Ca2+]i in either cell line. The oxotremorine-M-induced up-regulation of c-fos transcription was inhibited by 4-DAMP, but not by pirenzepine or AF-DX 116. Our findings thus suggest that ACh released from T-lymphocytes acts as an autocrine/paracrine factor, transmitting a Ca2+-dependent signal to the nuclei of T- and B-lymphocytes via M3 receptors.
Title: YM905, a novel M3 antagonist, inhibits Ca2+ signaling and c-fos gene expression mediated via muscarinic receptors in human T cells Fujii T, Kawashima K Ref: General Pharmacology, 35:71, 2000 : PubMed
Our earlier observations suggest that M3 muscarinic acetylcholine (ACh) receptors (mAChRs) are involved in Ca2+ signaling and regulation of c-fos gene expression in T lymphocytes. Here, we describe the effects of YM905, a novel M3 antagonist, on evoked Ca2+ signaling and c-fos gene expression in CEM human leukemic T cells. YM905 significantly inhibited increases in intracellular free Ca2+ evoked by 10 microM oxotremorine-M, an M1/M3 agonist (IC50=100 nM), and also inhibited 10 microM oxotremorine-M-induced upregulation of c-fos gene expression at 1 microM. These findings demonstrate that YM905 antagonizes the intracellular responses in T cells induced via mAChRs, possibly M3 receptors.
Title: Enhancement of cerebral cortical acetylcholine release by intraperitoneal acetic acid and its suppression by analgesics in freely moving rats Harada H, Hosonuma K, Fujii T, Kawashima K Ref: Neuroscience Letters, 284:163, 2000 : PubMed
Several lines of evidence suggest that central cholinergic neurons play a key role in the perception and control of pain. We investigated the effects of analgesics on the increase in central cholinergic activity and writhing responses elicited by i.p. injection of acetic acid. ACh efflux from the rat cerebral cortex and hippocampus was measured in the absence of a cholinesterase inhibitor using an in vivo microdialysis technique and a highly sensitive and specific radioimmunoassay. ACh efflux from the cerebral cortex was significantly increased during the first 30 min after acetic acid injection and then returned to the control levels. In contrast, acetic acid-induced writhing responses, indicative of the perception of pain, persisted for almost the entire 120 min observation period. No changes in ACh efflux were observed in the hippocampus. The centrally-acting analgesic morphine and the peripherally-acting analgesic indomethacin each completely abolished the enhanced cerebral cortical ACh efflux and the writhing, whereas diazepam, a muscle relaxant, selectively suppressed only the writhing. These results demonstrate that peripheral nociceptive stimulation transiently increases cholinergic activity in the cerebral cortex, but not in the hippocampus, and that analgesics suppress both the enhanced ACh efflux and the writhing induced by acetic acid.
Title: Extraneuronal cholinergic system in lymphocytes Kawashima K, Fujii T Ref: Pharmacol Ther, 86:29, 2000 : PubMed
Acetylcholine (ACh) is well known as a neurotransmitter in both the central and peripheral nervous systems in mammalian species. Both muscarinic and nicotinic ACh receptors have been identified in lymphocytes isolated from thymus, lymph node, spleen, and peripheral blood, and their stimulation by muscarinic and nicotinic agonists elicits a variety of functional and biochemical effects. On the basis of these findings, it has been postulated that the parasympathetic nervous system may play a role in immune-neurohumoral crosstalk. However, ACh present in the blood of several species has been localized to lymphocytes from various origins using radioimmunoassay. Moreover, using Northern blots or reverse transcription-polymerase chain reaction, expression of choline acetyltransferase, an ACh synthesizing enzyme, has been identified in human blood mononuclear leukocytes, human leukemic T-cell lines, and rat lymphocytes. Stimulation of T-lymphocytes with phytohemagglutinin activates the lymphoid cholinergic system, as evidenced by increased synthesis and release of ACh, increased acetylcholinesterase activity, and the increased expression of mRNA encoding choline acetyltransferase and ACh receptors. The observation that muscarinic receptor stimulation by ACh or agonists increases in [Ca(2)+](i) and up-regulates c-fos expression strongly argues that ACh synthesized and released from T-lymphocytes acts as an autocrine and/or paracrine factor regulating immune function. In summary, these data present a compelling picture in which immune function is not only regulated by the cytokine system, but is also under the control of an independent, lymphoid cholinergic system.
Title: Enhancement of the serotonin-mediated acetylcholine release by repeated desmethylimipramine treatment in the hippocampus of freely moving rats Fujii T, Ohba S, Nakai K, Fujimoto K, Suzuki T, Kawashima K Ref: Japanese Journal of Pharmacology, 80:303, 1999 : PubMed
A possible involvement of serotonin-mediated cholinergic activation in the antidepressant effect of desmethylimipramine (DMI) was investigated by determination of the effects of a single or repeated DMI administration on acetylcholine (ACh) release in the hippocampus using an in vivo microdialysis technique and a radioimmunoassay for ACh. Rats were administered DMI (10 mg/kg, i.p.) acutely or repeatedly for 21 days. A single or repeated DMI administration did not cause any significant effects on the basal ACh release compared with the respective controls. Atropine perfusion in the acutely DMI-treated or control rats increased the ACh release to the same degree. In repeatedly DMI-treated rats, serotonin (5-HT) (1 to 10 microM) perfusion enhanced significantly the ACh release. However, 5-HT in acutely DMI-treated rats enhanced significantly the ACh release only at 10 microM. 5-HT did not cause any changes in ACh release in control rats. Hippocampal 5-HT content of acutely DMI-treated rats was significantly higher than that of saline-treated control rats, while no difference was observed between the repeatedly DMI- and saline-treated rats. These findings suggest, for the first time, that DMI induced a facilitation of cholinergic neurotransmission in the rat hippocampus through the activation of 5-HT-receptor function.
Both muscarinic and nicotinic acetylcholine (ACh) receptors are known to be present on the surface of lymphocytes. We have shown that variable amounts of ACh are detectable in the blood of various mammals including humans, and a major portion of blood ACh is localized in circulating mononuclear leukocytes in humans. In order to investigate which types of blood cell are the source of ACh in human blood, expression of mRNA for choline acetyltransferase (ChAT, EC 2.3.1.6), which catalyzes ACh synthesis, was analyzed using human leukemic cell lines as models of lymphocytes and the reverse transcription-polymerase chain reaction (RT-PCR) method. We observed that mRNA for the same ChAT as that in the nervous system is expressed constitutively in all the T-cell lines tested, but not in B-, pre-lymphoma or monocytic cell lines. Furthermore, only T-cell lines showed high ACh-synthesizing activities and intracellular ACh contents. These results suggest that the major portion of ACh in the circulating blood originates from T-lymphocytes.
Title: Enhancement of cortical and hippocampal cholinergic neurotransmission through 5-HT1A receptor-mediated pathways by BAY x 3702 in freely moving rats Koyama T, Nakajima Y, Fujii T, Kawashima K Ref: Neuroscience Letters, 265:33, 1999 : PubMed
Involvement of serotonin (5-HT) in the regulation of cholinergic neuronal activity by modulation of acetylcholine (ACh) release has been reported for various regions of the brain. Cortical and hippocampal cholinergic neurotransmission is of particular importance in the mechanisms of attention as well as learning and memory. In the present study, we investigated the effect of R-(-)-2-[4-[(chroman-2-yl-methyl)-amino-butyl]-1,1-dioxo-benzo[d]++ +isothiazolone hydrochloride (BAY x 3702), a novel, high-affinity 5-HT1A receptor agonist, on ACh release in the cerebral cortex and hippocampus of freely moving rats using an in vivo microdialysis technique. Acetylcholine efflux from the cortex and hippocampus was measured every 30 m using a sensitive and specific radioimmunoassay and was stable for at least 5 h. The ACh efflux from the cortex and hippocampus was increased significantly by BAY x 3702 (0.3 mg/kg, i.p.) compared with saline administration. WAY-100635 (0.6 mg/kg, s.c.), a selective 5-HT1A receptor antagonist, eliminated the augmentation of ACh efflux induced by BAY x 3702 in both brain regions. These results demonstrate that stimulation by BAY x 3702 enhanced ACh release in both the cortex and hippocampus through 5-HT1A receptor-mediated pathways.
Title: Nerve growth factor increases the synthesis and release of acetylcholine and the expression of vesicular acetylcholine transporter in primary cultured rat embryonic septal cells Oosawa H, Fujii T, Kawashima K Ref: Journal of Neuroscience Research, 57:381, 1999 : PubMed
Acetylcholine (ACh) is synthesized by choline acetyltransferase (ChAT) in the cytoplasm of cholinergic nerve terminals and transported into synaptic vesicles by vesicular ACh transporter (VAChT). The genes encoding ChAT and VAChT are colocalized within the genome, and their products are known to be coregulated by various neurotrophic factors. In the present study, nerve growth factor (NGF; 100 ng/ml) was shown to enhance expression of VAChT and ChAT mRNA in primary cultured rat embryonic septal cells. By using a radioimmunoassay, we also found that NGF increased both neuronal content and spontaneous release of ACh, which were first detected on day 2 of culture and time-dependently increased up to day 10. Stimulated release of ACh elicited by high K+ (50 mM KCl) was also significantly greater in NGF-treated cells than in control cells. NGF enhanced immunoreactivity to antiserum against VAChT, indicating that the augmented responses were due to, at least in part, increased expression of VAChT protein. In contrast, the numbers of immunocytochemically positive cells were unaffected. Thus, NGF appears to augment ACh synthesis, its transport into synaptic vesicles, and its subsequent release. The data also suggest that NGF facilitates growth and development of cholinergic neurons, but not their survival.
Human amniotic epithelial cells express makers of glial and neural stem cells. These cells also synthesize and release acetylcholine and catecholamines. This study of amniotic fluid demonstrated that acetylcholine and catecholamines can be readily identified in the fluid. Norepinephrine was the major catecholamine present, although dopamine and DOPAC could also be detected. The physiologic role of these amniotic fluid neurotransmitters in fetal-placental interactions and nervous system development is currently under investigation.
Title: Diversity of mRNA expression for muscarinic acetylcholine receptor subtypes and neuronal nicotinic acetylcholine receptor subunits in human mononuclear leukocytes and leukemic cell lines Sato KZ, Fujii T, Watanabe Y, Yamada S, Ando T, Kazuko F, Kawashima K Ref: Neuroscience Letters, 266:17, 1999 : PubMed
Previously, we reported that various levels of acetylcholine (ACh), currently known as a neurotransmitter, are detectable in the blood of several mammals including humans and that most blood ACh originates from T-lymphocytes. To investigate whether ACh in the blood acts on lymphocytes and participates in the modulation of immune responses, we have analyzed the expression of mRNA for muscarinic (Ms) ACh receptor subtypes and nicotinic (Nc) ACh receptor subunits using reverse transcription-polymerase chain reaction (RT-PCR) methods. The cells tested were human peripheral mononuclear leukocytes (MNLs) from seven healthy donors and eight leukemic cell lines, as models of lymphocytes. We detected mRNA expression for various neuronal Nc receptor subunits and Ms receptor subtypes in all of the MNL samples and in all of the cell lines tested. However, the expression pattern of mRNA for neuronal Nc receptor subunits (alpha2-alpha7 and beta2-beta4) and Ms receptor subtypes (m1-m5) varied among the individuals and cell lines. No expression of mRNA for three muscle-type Nc receptor subunits (alpha1, beta1 and epsilon) was observed in the MNLs and cell lines. These results indicate that both neuronal-type Nc and Ms ACh receptors are present on the surface of lymphocytes.
The induction of mRNA for choline acetyltransferase (ChAT), which catalyzes acetylcholine (ACh) synthesis was investigated in human mononuclear leukocytes (MNL) stimulated by phytohemagglutinin (PHA), a T-cell activator, using the reverse transcription-polymerase chain reaction. Stimulation of MNL by PHA induced the expression of ChAT mRNA, and potentiated ACh synthesis. ChAT mRNA induction required more time than the induction of interleukin-2 mRNA. Expression of the gene encoding the vesicular ACh transporter, which mediates ACh transport in cholinergic neurons, was not observed in PHA-stimulated MNL, suggesting that the mechanisms controlling ACh release from T-lymphocytes differ from those in cholinergic neurons. These findings demonstrate that activation of T-lymphocytes up-regulates ACh synthesis in the blood, and suggest that ACh plays an important role as a neuroimmunomodulator besides its role as a neurotransmitter.
Title: Acetylcholine synthesis and muscarinic receptor subtype mRNA expression in T-lymphocytes Kawashima K, Fujii T, Watanabe Y, Misawa H Ref: Life Sciences, 62:1701, 1998 : PubMed
We used a sensitive and specific radioimmunoassay for acetylcholine (ACh), and detected significant amounts of ACh in the blood of various mammals, including humans. About 60% of human blood ACh was localized in mononuclear leukocytes. Human leukemic T-cell lines, used as T-lymphocyte models, contained both ACh and choline acetyltransferase (ChAT) activity. Furthermore, ChAT mRNA and protein were detected in the T-cell line MOLT-3. Phytohemagglutinin, a T-cell activator, increased both synthesis and release of ACh by MOLT-3 cells. Muscarinic receptor subtype mRNA expression was confirmed in various T-cell lines. These findings indicate that ACh synthesized by ChAT in T-lymphocytes acts on the muscarinic receptors on lymphocytes in autocrine and/or paracrine pathways and suggest that ACh in blood functions as a modulator of T-cell-dependent immune responses.
Title: Effect of WAY-100135 on the hippocampal acetylcholine release potentiated by 8-OH-DPAT, a serotonin1A receptor agonist, in normal and p-chlorophenylalanine-treated rats as measured by in vivo microdialysis Nakai K, Fujii T, Fujimoto K, Suzuki T, Kawashima K Ref: Neurosci Res, 31:23, 1998 : PubMed
The mechanisms involved in the enhancement of acetylcholine (ACh) release in the rat hippocampus by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a serotonin (5-HT)1A receptor agonist, were investigated using in vivo microdialysis. Administration of p-chlorophenylalanine (PCPA, 300 mg/kg, i.p.), a tryptophan hydroxylase inhibitor, 3 days before the dialysis experiments reduced the hippocampal 5-HT content to 30% of that in saline-treated rats, but did not affect basal ACh release in the hippocampus. 8-OH-DPAT administered systemically (0.5 mg/kg, s.c.) or applied locally (30 microM) into the hippocampus through the dialysis probe significantly enhanced the release of ACh in the hippocampus of PCPA-treated rats to the same degree as that in saline-treated rats. Pretreatment with (+)WAY-100135 (5 mg/kg, i.p.), a selective 5-HT1A receptor antagonist, completely eliminated the enhancement of ACh release induced by locally applied 8-OH-DPAT, but only partially reduced the effects induced by systemically administered 8-OH-DPAT, in both groups of rats. Systemically administered 8-OH-DPAT induced hyperlocomotion in the both saline- and PCPA-treated rats, but this was not eliminated by (+)WAY-100135. 8-OH-DPAT applied locally into the hippocampus did not elicit hyperlocomotion in either group of rats. These results suggest that the modification of endogenous 5-HT release via the 5-HT1A autoreceptor is not involved in the 8-OH-DPAT-induced increase of hippocampal ACh release, and that the increase of ACh release induced by locally applied 8-OH-DPAT involves mainly hippocampal postsynaptic 5-HT1A receptor stimulation. In addition, a possibility that subtypes of 5-HT receptors other than the 5-HT1A receptor, probably 5-HT7 receptor in the septum as well as postsynaptic 5-HT1A receptor in the hippocampus, are involved in the increased hippocampal ACh release induced by systemically administered 8-OH-DPAT is discussed.
Title: Cloning and sequencing of a gene cluster for the meta-cleavage pathway of aniline degradation in Acinetobacter sp. strain YAA Takeo M, Fujii T, Takenaka K, Maeda Y Ref: J.Ferment.Bioeng, 85:514, 1998 : PubMed
Acinetobacter sp. strain YAA is able to use aniline and o-toluidine as the sole carbon and energy source. This strain has several different plasmids and acridine orange curing suggested that aniline utilization in strain YAA was plasmid-encoded. The gene cluster involved in aniline oxidation was cloned in Escherichia coli JM109 from the total plasmid DNA of strain YAA. A recombinant E. coli containing an 18.5 kb insert fragment showed yellow colouration on aniline-containing plates, indicating the formation of 2-hydroxymuconic semialdehyde from aniline. In addition, subcloning of a 9.0 kb SalI fragment from the insert in E. coli resulted in the accumulation of catechol. Southern hybridization studies indicated that the aniline oxygenase gene (atdA) was present on one of the plasmids, pYA1. These results suggest that in strain YAA aniline is degraded via catechol through a pathway involving meta-cleavage of the benzene-ring by plasmid-encoded genes including atdA.
Title: Maintenance of constant blood acetylcholine content before and after feeding in young chimpanzees Fujii T, Mori Y, Tominaga T, Hayasaka I, Kawashima K Ref: Neuroscience Letters, 227:21, 1997 : PubMed
We have shown that acetylcholine (ACh) is present in the blood of various species of mammals using a specific, sensitive radioimmunoassay. In the present study, the effect on blood and plasma ACh levels of feeding after overnight fasting was studied in one male and five female 4- to 7-year-old chimpanzees. The mean basal ACh concentrations of the blood and plasma were 3143 +/- 380 and 184 +/- 10 pg/ml (+/-SEM, n = 6), respectively. Feeding each chimpanzee 500 g boiled sweet potatoes as breakfast at 1000 h and tap water given ad libitum did not affect the ACh content of the blood and plasma, and constant values of the blood and plasma ACh contents were observed for 4 h after the feeding. Hematocrit and plasma acetylcholinesterase (AChE) activity were also insensitive to feeding. No correlation was observed between plasma AChE activity and either blood or plasma ACh content. The results of the present study indicate that the blood ACh of chimpanzees is distributed mainly in the blood cell fraction, and that the blood ACh content is not regulated directly by cholinergic nerve activity or by plasma AChE activity.
Title: Demonstration of the facilitatory role of 8-OH-DPAT on cholinergic transmission in the rat hippocampus using in vivo microdialysis Fujii T, Yoshizawa M, Nakai K, Fujimoto K, Suzuki T, Kawashima K Ref: Brain Research, 761:244, 1997 : PubMed
The role of the serotonin (5-HT)1A receptor in the regulation of acetylcholine (ACh) release in the hippocampus was investigated using an in vivo microdialysis technique and a sensitive radioimmunoassay specific for ACh. The mean (+/- S.E.M.) basal ACh contents in the hippocampal perfusate of conscious, freely moving rats was 60 +/- 4 (n = 29) and 3691 +/- 265 fmol/30 min (n = 31), respectively, in the absence and presence of physostigmine (Phy) in the perfusion fluid. Systemic administration of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 0.5 mg/kg, s.c.), a 5-HT1A agonist, significantly enhanced ACh release both in the presence and absence of Phy. Local application of 8-OH-DPAT (3-30 microM) into the hippocampus through the microdialysis probe significantly potentiated ACh release only in the presence of Phy, whereas no significant effect was observed in its absence. Pretreatment with NAN-190 (3 mg/kg, i.p.), a 5-HT1A antagonist, eliminated the increasing effect of systemically applied 8-OH-DPAT on ACh release, while NAN-190 alone had no effect on basal ACh release either in the absence or presence of Phy. Consistent with the time course of ACh release, systemic administration of 8-OH-DPAT evoked hyperlocomotion, which was reversed by NAN-190. However, local hippocampal application of 8-OH-DPAT did not affect the locomotor activity of the rats. These findings suggest that at least two different sites are involved in the 8-OH-DPAT-induced increase in the release of ACh in the rat hippocampus in vivo.
Title: Low sensitivity of adrenal chromaffin cells to acetylcholine, neostigmine and oxotremorine in 21-day-old rats Fujino Y, Fujii T Ref: Japanese Journal of Pharmacology, 75:145, 1997 : PubMed
In the previous study, we have demonstrated that nicotine hardly induces catecholamine release from adrenal medulla of 21-day-old rats. The present study examined the responsiveness of the adrenal chromaffin cells to acetylcholine in vitro and neostigmine and oxotremorine in vivo in 21-day-old and 8-week-old rats. As assessed by electron microscopy, the number of the chromaffin granules was markedly decreased and the content of adrenaline in adrenals was diminished significantly by oxotremorine treatment in 8-week-old rats, whereas these changes did not occur in 21-day-old rats. Morphological changes of the adrenal chromaffin cells, with respect to exocytosis, were not observed in neostigmine-treated 21-day-old rats and acetylcholine-treated adrenal slices prepared from 21-day-old rats. Catecholamine release was hardly evoked by acetylcholine in these slices as judged by measuring the catecholamine content in the medium. These results indicate that the sensitivity of the chromaffin cells to these secretagogues in 21-day-old rats is very low when compared to that in young adult rats.
Title: Nitric oxide increases stimulation-evoked acetylcholine release from rat hippocampal slices by a cyclic GMP-independent mechanism Suzuki T, Nakajima K, Fujimoto K, Fujii T, Kawashima K Ref: Brain Research, 760:158, 1997 : PubMed
Nitric oxide (NO) is an endothelium-derived relaxing factor and its main mechanism of action is activation of soluble guanylyl cyclase. NO and NO-related compounds have been reported to affect several neuronal functions in the central nervous system. In this study, we investigated the effects of NO donors (sodium nitroprusside (SNP) and (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK409)) on acetylcholine (ACh) release from rat hippocampal slices. SNP (10(-5) M) and FK409 (10(-4) M) increased electrical stimulation-evoked ACh release without affecting basal release. As dibutyryl cyclic GMP inhibited stimulation-evoked ACh release, the effects of these NO donors were not due to soluble guanylyl cyclase activation. Atropine increased stimulation-evoked ACh release by blocking presynaptic muscarinic autoreceptors, and SNP increased stimulation-evoked ACh release in the presence of atropine, suggesting that SNP and atropine increase stimulation-evoked ACh release by different mechanisms. The present results indicate that NO enhances some part of the excitation-secretion coupling pathway without inducing ACh release directly and these effects are mediated by cyclic GMP-independent mechanism.
Title: Oral administration of KW-5092, a novel gastroprokinetic agent with acetylcholinesterase inhibitory and acetylcholine release enhancing activities, causes a dose-dependent increase in the blood acetylcholine content of beagle dogs Yamada S, Fujii T, Kawashima K Ref: Neuroscience Letters, 225:25, 1997 : PubMed
Acetylcholine (ACh) was detected in the blood and plasma of beagle dogs using a specific, sensitive radioimmunoassay. The mean basal ACh contents in the blood and plasma of beagle dogs were 451 +/- 65 and 83.5 +/- 12.3 pg/ml (+/- SEM, n = 7), respectively, and were lower than the contents in humans reported previously by our laboratory. Oral administration of KW-5092 (10-30 mg/kg), a gastroprokinetic agent with acetylcholinesterase (AChE) inhibitory and ACh release enhancing activities, caused a dose-dependent increase in the ACh content of both the blood and plasma, as well as several behavioral side effects due to peripheral cholinergic stimulation. The size of the increase in the plasma ACh content at each dose of KW-5092 was greater than that in the blood, indicating that KW-5092 caused the increase in the blood ACh content through elevation of the plasma ACh content, by inhibition of AChE and facilitation of ACh release. These results demonstrate that the blood ACh of beagle dogs is present mainly in the blood cells and to a lesser degree in the plasma, and that KW-5092 increased the blood ACh content mainly by increasing the plasma ACh concentration.
In order to clarify the origin of acetylcholine (ACh) in human blood, we measured the content and synthesis activity of ACh in several human leukemic cell lines. The intracellular ACh content determined by a specific and sensitive radioimmunoassay in the human leukemic T cell lines, HSB-2, MOLT-3, and CEM, was 79.6, 36.2, and 9.5 pmol/10(6) cells, respectively. These values were 9-70-fold higher than those of other cell lines, including a helper T cell line, Jurkat. Stimulation of HSB-2 and MOLT-3 by phytohemagglutinin (PHA) increased both the intracellular content and release of ACh into the culture medium, but did not influence the intracellular content and release of ACh in CEM. ACh synthesis activity was found in all the T cell lines tested. Bromoacetylcholine (100 microM), a choline acetyltransferase (ChAT) inhibitor, and bromoacetyl-L-carnitine (100 microM), a carnitine acetyltransferase (CarAT) inhibitor, decreased ACh-synthesizing activity in MOLT-3, and HSB-2 and CEM, by about 50% and 30%, respectively, indicating that both ChAT, and to a lesser extent CarAt, are involved in ACh synthesis in T cells. These results suggest that T lymphocytes have the potential to synthesize and release ACh, which may play a role in regulating T cell-dependent immune responses.
Title: Crystal structure of L-2-haloacid dehalogenase from Pseudomonas sp. YL. An alpha/beta hydrolase structure that is different from the alpha/beta hydrolase fold Hisano T, Hata Y, Fujii T, Liu JQ, Kurihara T, Esaki N, Soda K Ref: Journal of Biological Chemistry, 271:20322, 1996 : PubMed
L-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of L-2-haloalkanoic acids to yield the corresponding D-2-hydroxyalkanoic acids. The crystal structure of the homodimeric enzyme from Pseudomonas sp. YL has been determined by a multiple isomorphous replacement method and refined at 2.5 A resolution to a crystallographic R-factor of 19.5%. The subunit consists of two structurally distinct domains: the core domain and the subdomain. The core domain has an alpha/beta structure formed by a six-stranded parallel beta-sheet flanked by five alpha-helices. The subdomain inserted into the core domain has a four helix bundle structure providing the greater part of the interface for dimer formation. There is an active site cavity between the domains. An experimentally identified nucleophilic residue, Asp-10, is located on a loop following the amino-terminal beta-strand in the core domain, and other functional residues, Thr-14, Arg-41, Ser-118, Lys-151, Tyr-157, Ser-175, Asn-177, and Asp-180, detected by a site-directed mutagenesis experiment, are arranged around the nucleophile in the active site. Although the enzyme is an alpha/beta-type hydrolase, it does not belong to the alpha/beta hydrolase fold family, from the viewpoint of the topological feature and the position of the nucleophile.
Various concentrations of acetylcholine (ACh) were detected in samples of bovine, goat, horse, porcine, rat and sheep blood and plasma using a specific, sensitive radioimmunoassay. The ACh levels in whole blood in bovine and horse samples were about 40- and ten-fold higher, respectively, than in humans, but levels comparable to those in humans were measured in porcine samples. Goat, rat and sheep samples had lower whole blood ACh concentrations than those of humans. When plasma samples were assayed, the ACh contents of bovine and porcine plasma were found to be about two- to five-fold those of human. On the other hand, levels in horse, goat, rat and sheep samples were much lower than in humans. The ratio of the ACh contents of plasma to whole blood was high in porcine and rat samples, indicating that porcine and rat blood ACh is distributed mostly in the plasma, while in the other species tested most of the ACh is present in the blood cells. These results demonstrate that variable levels of ACh are present in the blood of different species, and that the distribution of ACh in the blood constituents varies according to species.
Title: Effects of the centrally acting cholinesterase inhibitors tetrahydroaminoacridine and E2020 on the basal concentration of extracellular acetylcholine in the hippocampus of freely moving rats Kawashima K, Sato A, Yoshizawa M, Fujii T, Fujimoto K, Suzuki T Ref: Naunyn Schmiedebergs Arch Pharmacol, 350:523, 1994 : PubMed
The effects of the centrally acting cholinesterase (ChE) inhibitors, tetrahydroaminoacridine (THA) and E2020 (1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl] methylpiperidine hydrochloride), potential drugs for the treatment of senile dementia, on the basal extracellular acetylcholine (ACh) concentration in the hippocampus of freely moving rats, were determined using a microdialysis technique without the use of a ChE inhibitor in the perfusion fluid and a sensitive RIA. The mean (+/- SEM) basal ACh content in the perfusate was 103.1 +/- 3.6 fmol/sample collected over 30 min when microdialysis probes with a length of 3 mm dialysis membrane were used. The content of ACh decreased to an almost undetectable level upon perfusion of magnesium, suggesting that, in the present study, most of the ACh detected in the perfusates was due to cholinergic neuronal activity. THA (1.65 mg/kg, i.p.) produced an insignificant increase in the extracellular ACh concentration, but a dose of 5 mg/kg, i.p. caused a prolonged and significant 5.5-fold increase from the control value. E2020 (0.65 and 2 mg/kg, i.p.) produced significant, prolonged and dose-dependent increases (4 and 12 times the control value, respectively), the peak effect occurring within 1 h. Perfusion with 10 mumol/l physostigmine produced an about 30-fold increase of ACh output, suggesting that the basal extracellular ACh concentration is highly dependent on ChE activity. When ChE was inhibited locally by perfusion with physostigmine, THA (5 mg/kg) produced a transient and, at its maximum, a 1.42-fold increase in extracellular ACh concentration.
